Not stated.

abnormality is only one step (first hit) in the progression of malignant clone. In order to gain growth advantage, the transforming cells need other molecular changes within the cells and in the marrow microenvironment (second hit). The cases who attained spontaneous remission suggest that the development of cytogenetic abnormality might have not been supported by the other cellular and microenvironmental changes [65, 66]. Additionally, the mutation may have developed in a hematopoietic cell with limited self-renewal capacity and may not have involved the whole stem cell pool [66]. Two cases of Bader-Meunier et al. [68] suggest that patients with MDS who do not show any chromosomal abnormality can also achieve complete remission (**Table 9**). These cases show that patients who are stable should be closely observed

**Genetic analysis Management of**

45,XY,−7 [12] Transfusions <2 years

46,XY [13]/45,XY, −7 [7] No 8 months

ivIgG and transfusion

/BMT planned

AZA stopped, transfusions

NS# BMT planned

NS# BMT planned

**therapy** 

**Outcome /time elapsed to hematologic /cytogenetic complete remission (hem/cyto rem) (months)**

(hem + cyto rem)

(hem + cyto rem)

(hem + cyto rem, hem rem partial)

4 weeks (hem recovery; cyto rem unknown©)

13 months (hem + cyto rem)

17 months (hem + cyto rem)

10 months (hem + cyto rem)

12 months (hem + cyto rem)

No 5 months

**Duration of follow-up after cyto rem attained (months)**

108 months [61]

14 months [61]

95 months [61]

67 months [61]

21 months [62]

NS# [63]

NS# [64]

NS# [64]

**Reference**

for some time before potentially toxic therapies are started.

**Cases Diagnosis Age/**

74 Myelodysplastic Syndromes

1 De novo MDS†

2 De novo MDS†

3 t-MDS†

4 t-MDS†

6 t-MDS†

8 t-MDS†

 (after completing rabdomyosarcoma therapy)

 (after completing medulloblastoma therapy)

5 RAEB 14/12,

(Spina bifida, ESRF\*

7 t-MDS†\*\*\* after completing Hodgkin's disease therapy

 ; AZA therapy\*\*

 (after completing Ewing sarcoma therapy

**sex** 

8/12, M

15/12, M

10.5, M

M

46,XY,del (7)(q22q32) [4] (out of 40 metaphases)

8/M 46 XY,del(7)(q31q36) [5]/46, XY,+der(1)t(1;7)(p11.2;p11.2), -(3)/46,XY,+dic(1;21) (p11.2;p11.2),

19, F 45,XY, −7 [5] (out of 62 metaphases)

15, F 45, XY, −7 [19] (out of 20 metaphases)

19, M 46,XY,(11;16)(q23;p13.3) [13] (out of 20 etaphases)

−21 [4] and other anomalies

46,XY [13]/45,XY, −7 [7] Transfusions

\*\*\*The patient had pancytopenia persisting after the 1st course of Hodgkin's disease therapy.

**Table 9.** Patients with MDS who achieved remission spontaneously, after supportive or vitamin B12 and folic acid therapy [61–70].

#### **4.2. Vitamin B12 (VB12) and folic acid deficiency and transient chromosome abnormalities which mimick MDS**

Vitamin B12 and folic acid deficiencies can present as mono-, bi-, and pancytopenia [106], myelodysplasia, genetic abnormalities like increased frequency of spontaneous chromosome breakage and centromere spreading [107, 108], elongation and despiralization of chromosomes [107], multiple rearrangements and deletions of different chromosomes [108] which can mimick that of MDS (**Table 9**, patient 11) [67].

Many of these chromosomal abnormalities were reported to reduce [107], completely disap‐ pear [67, 107, 108] or persist up to 6–12 months after hematological remission was attained [107] after therapy.

These cases show that defective synthesis and repair of DNA which were reversed by VB12 and folate plays role in the pathogenesis of genetic abnormalities in megaloblastic anemia.

Increased immature myeloid cells indistinguishable from myeloblasts in the bone marrow of patients with vitamin B12 and folate deficiency make the differentiation between megaloblastic anemia and MDS more difficult [109].

On the other hand, in our clinics, we have encountered considerable number of patients who had VB12 and/or folic acid deficiency coexistent with MDS or leukemia.

#### **5. Mutations in the elderly and other cases**

In more than 10% of healthy people older than 70 years, clonal hematopoiesis is present [10]. Additionally, loss of Y chromosome in hematopoietic cells in association with aging was also reported [1]. Therefore, clinicians should keep reserved when abnormalities in molecular genetics and karyotype are found in the elderly when dysplasia is absent [10].

Mutations like del(20q), +8, −Y have been reported in patients with aplastic anemia or other cytopenic syndromes who were good responders to immunosuppressive therapy and/or no evidence of MDS findings in the follow-up [1].

#### **6. Acute myeloblastic leukemia**

#### **6.1. AML with low blast cell count**

AML is distinguished from MDS by the percentage of the blasts which is higher than 30% in AML in children (>20% in adults) and lower than 30% (lower than 20% in adults) in MDS. However, patients that have blast cells lower than 30%, but cytogenetic features characteristic of childhood de novo AML [t(8;21), inv(16), t(11;17), t(9;11), i(1)] is designated as AML with low blast cell count (AML-LBC).

Those with AML-LBC were significantly younger than MDS cases (3.7 vs 7.4); their dysplasia score was lower and the response to AML type chemotherapy was higher than that of MDS patients. The authors encountered chloroma only in AML-LBC [71].

#### **6.2. AML with myelodysplasia-related changes (AML-MRC)**

**4.2. Vitamin B12 (VB12) and folic acid deficiency and transient chromosome abnormalities**

Vitamin B12 and folic acid deficiencies can present as mono-, bi-, and pancytopenia [106], myelodysplasia, genetic abnormalities like increased frequency of spontaneous chromosome breakage and centromere spreading [107, 108], elongation and despiralization of chromosomes [107], multiple rearrangements and deletions of different chromosomes [108] which can

Many of these chromosomal abnormalities were reported to reduce [107], completely disap‐ pear [67, 107, 108] or persist up to 6–12 months after hematological remission was attained

These cases show that defective synthesis and repair of DNA which were reversed by VB12 and folate plays role in the pathogenesis of genetic abnormalities in megaloblastic anemia.

Increased immature myeloid cells indistinguishable from myeloblasts in the bone marrow of patients with vitamin B12 and folate deficiency make the differentiation between megaloblastic

On the other hand, in our clinics, we have encountered considerable number of patients who

In more than 10% of healthy people older than 70 years, clonal hematopoiesis is present [10]. Additionally, loss of Y chromosome in hematopoietic cells in association with aging was also reported [1]. Therefore, clinicians should keep reserved when abnormalities in molecular

Mutations like del(20q), +8, −Y have been reported in patients with aplastic anemia or other cytopenic syndromes who were good responders to immunosuppressive therapy and/or no

AML is distinguished from MDS by the percentage of the blasts which is higher than 30% in AML in children (>20% in adults) and lower than 30% (lower than 20% in adults) in MDS. However, patients that have blast cells lower than 30%, but cytogenetic features characteristic of childhood de novo AML [t(8;21), inv(16), t(11;17), t(9;11), i(1)] is designated as AML with

had VB12 and/or folic acid deficiency coexistent with MDS or leukemia.

genetics and karyotype are found in the elderly when dysplasia is absent [10].

**5. Mutations in the elderly and other cases**

evidence of MDS findings in the follow-up [1].

**6. Acute myeloblastic leukemia**

**6.1. AML with low blast cell count**

low blast cell count (AML-LBC).

**which mimick MDS**

76 Myelodysplastic Syndromes

[107] after therapy.

mimick that of MDS (**Table 9**, patient 11) [67].

anemia and MDS more difficult [109].

In a subgroup of leukemia that was introduced by WHO is AML with myelodysplasia-related changes (AML-MRC) which defines AML arising from previous MDS or MDS/MPN, with an MDS-related cytogenetic abnormality and/or AML with multilineage dysplasia (AML-MLD). This group was reported to have worse overall survival when compared with patients with AML-not otherwise specified [110].

#### **6.3. MDS with myelofibrosis, AML-M7, and other disorders**

The blast cell percentage in the bone marrow (>30% in children and >20% in adults) and t(1;22) (p13;q13) differentiated AML-M7 from MDS.

Although hypoplastic MDS with increased reticulin ± collagen fibers is rare in childhood [4], hypoplastic myelofibrosis may be encountered in childhood [111].

### **7. Idiopathic cytopenia of undetermined significance (ICUS) and idiopathic dysplasia of undetermined significance (IDUS)**

Patients with persistent (marked constant) cytopenia(s) involving one of more hematopoietic lineages, for at least 6 months, in the setting of absent multiunilineage dysplasia and cytoge‐ netic abnormality except –Y, +8, del(20q) were suggested to be termed as idiopathic cytopenia of undetermined significance (ICUS) [5, 58, 112–116]. Criteria of cytopenia for diagnosis of both MDS and ICUS according to the 2007 Consensus Group are: Hemoglobin (Hb) <11g/dl and/or neutrophils <1500/mm3 , and/or thrombocytes <100,000/mm3 . The cut off levels for Hb is 10 g/dl, for neutrophils 1800/mm3 according to WHO and International Working Group on Morphology of MDS (IWGM-MDS) [58, 114].

The terms "ICUS-anemia, ICUS-neutropenia, ICUS-thrombocytopenia, ICUS-bicytopenia, or bi/pancytopenia" were also proposed in which the cut-off levels of cytopenia were the same of those in 2007 Consensus Group, except that of neutropenia which was proposed as <1000/mm3 [114].

For patients with morphological dysplasia (>10% in a major cell line) with/without karyotypic abnormalities but no or mild cytopenia, idiopathic dysplasia of undetermined significance (IDUS) has been proposed [5, 7]. Criteria of mild cytopenia has been reported as Hb ≥ 11 g/dl, neutrophils ≥ 1500/mm<sup>3</sup> , thrombocytes ≥ 100,000/mm<sup>3</sup> [113] (**Table 10**).

Patients with both ICUS and IDUS may progress to overt MDS, MPN, MDS/MPN overlap disease, chronic myelomonocytic leukemia, AML after a variable period. ICUS can also reportedly transform to systemic mastocytosis, non-Hodgkin lymphoma, aplastic anemia [5, 113, 114]. There is no proof that every patient with ICUS or IDUS will develop neoplasia [114]. ICUS was reported to resolve spontaneously also [112]. However, the median overall survival in ICUS group was reported 44.3 months, being shorter than RA but longer than RCMD [116].


\* Cytopenia in one or more of hematopoietic lineages lasting for at least 6 months, with Hb <11 g/dl and/or neutrophils <1500/mm3 , and/or thrombocytes <100,000/mm3 .

\*\*Cocriteria of MDS: Colony forming cells and reticulocytes in circulation, abnormal immunophenotyping by flow cytometry, monoclonality of myeloid cells detected by molecular markers or mutations, abnormal gene expression profile by mRNA profiling assays.

† If one or more cocriteria are found, the disorder should be called 'highly susceptive for a clonal myeloid disease/MDS' [113].

**Table 10.** Diagnostic criteria for ICUS and IDUS [5, 58, 112–116].

On the other hand, recent reports demonstrated that ICUS had a broad spectrum including patients with both nonclonal and clonal hematopoiesis, the latter being called as clonal hematopoiesis of indeterminate potential (CHIP) [112]. Hence, 35% of ICUS patients were found to carry a somatic mutation or chromosomal abnormality indicative of clonal hemato‐ poiesis [117] called clonal ICUS (CCUS).

Differentiation between MDS and ICUS may be challenging. In ICUS, FISH may reveal a very small clone carrying MDS-related cytogenetic defect [113, 114]. Clonal expansion of such a small clone in ICUS and occurrence of slight cytopenia in IDUS in the follow-up, points at an imminent transformation to MDS [58, 113, 114].

These patients should be examined regularly, like in low-risk MDS, from the aspect of hematological findings, karyotype, FISH, flow cytometry, and flow FISH, if available [114].

Reduced number of colony forming unit (CFU) progenitor cells like CFU-granulocytemacrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) show impaired bone marrow function in MDS [58]. While BFU-E is markedly reduced in MDS, it is reduced only in a minority of patients with ICUS and IDUS [114]. However, reduced numbers of CFU-GM and BFU-E are also found in aplastic anemia, acute leukemia, and in post chemotherapy conditions, but not in nonclonal cytopenias like vitamin B12 deficiency, autoimmune hemo‐ lytic anemia and chronic inflammation [58] (**Table 10**).

Screening for molecular lesions by exome sequencing and other "omics-based techniques" can be used, in order to search clonality. However, these techniques are expensive and not practical [58]. Human androgen receptor gene-based assay (HUMARA) which is promising has restrictions, since it can be used only in females and it is positive in other clonal disorders also [58, 116]. Application of flow cytometric tools are of considerable help [58] (see Section 10). Patients with CCUS can be differentiated from low-risk MDS only by lack of dysplasia [112].

The data about patients with ICUS, CCUS, and IDUS are limited. Future studies will enlighten the pathogenesis of ICUS and IDUS which is not well understood yet.

#### **8. Autoimmune disorders**

113, 114]. There is no proof that every patient with ICUS or IDUS will develop neoplasia [114]. ICUS was reported to resolve spontaneously also [112]. However, the median overall survival in ICUS group was reported 44.3 months, being shorter than RA but longer than RCMD [116].

**Idiopathic dysplasia of unknown**

**significance (IDUS)**

Present in a minority [112]

–?

**Idiopathic cytopenia of unknown**

Constant marked cytopenia\* Present [5, 58, 112–116] Absent [5, 58, 112–116] Diagnostic criteria of MDS Absent [5, 58, 112–116] Absent [5, 58, 112–116]

† Absent [113]; flow cytometric abnormalities:

clone carrying MDS-related cytogenetic defect

No [113] –

Marked reduction in BFU-E In a minority of patients [112] In a minority of patients [112] Ring sideroblasts >15% Absent [114] In a majority of patients [114]

Cytopenia in one or more of hematopoietic lineages lasting for at least 6 months, with Hb <11 g/dl and/or neutrophils

If one or more cocriteria are found, the disorder should be called 'highly susceptive for a clonal myeloid disease/MDS'

On the other hand, recent reports demonstrated that ICUS had a broad spectrum including patients with both nonclonal and clonal hematopoiesis, the latter being called as clonal hematopoiesis of indeterminate potential (CHIP) [112]. Hence, 35% of ICUS patients were found to carry a somatic mutation or chromosomal abnormality indicative of clonal hemato‐

Differentiation between MDS and ICUS may be challenging. In ICUS, FISH may reveal a very small clone carrying MDS-related cytogenetic defect [113, 114]. Clonal expansion of such a

\*\*Cocriteria of MDS: Colony forming cells and reticulocytes in circulation, abnormal immunophenotyping by flow cytometry, monoclonality of myeloid cells detected by molecular markers or mutations, abnormal gene expression

– No [113]

Dysplasia (>10% of cells) Absent [112–115] Present [112–116]

Clonality by HUMARA In a minority of patients [112] (CCUS) Not known [112]

Age Older [113] Younger [113] Erythropoietin level Low [113] Adequate [113]

.

**significance (ICUS)**

not known [112]

Karyotype typical for MDS Absent but FISH may reveal a very small

[113, 114]

, and/or thrombocytes <100,000/mm3

**Table 10.** Diagnostic criteria for ICUS and IDUS [5, 58, 112–116].

poiesis [117] called clonal ICUS (CCUS).

Diagnostic cocriteria of MDS\*\*,

78 Myelodysplastic Syndromes

Other diseases leading to

Other diseases leading to

profile by mRNA profiling assays.

cytopenia

dysplasia

\*

†

[113].

<1500/mm3

Patients with chronic immune stimulation and autoimmune disorders have a tendency to develop malignant neoplasias and MDS.

On the other hand, MDS and AML can trigger paraneoplastic syndromes and manifestations including inflammatory paraneoplasia, like seronegative rheumatoid arthritis, Sweets syndrome, hemophagocytic lymphohistiocytosis, pyoderma gangraenosum, cutaneous vasculitis, lupus-like symptoms [72, 73], polychondritis [73], Behçet syndrome, inflammatory bowel disease, cryoglobulins, vitiligo, autoimmune hemolytic anemia, peripheral neuropathy, by 12–19% in adults but in lower frequency in childhood [74].

Relevance of autoimmune disorders with prognosis of MDS is disputable. Their response to immunosuppressive therapy is good [72].

In MDS, not only the stem cells but an inflammatory microenvironment is also involved. Therefore, the inflammatory microenvironment aggravates ineffective hematopoiesis and carcinogenesis/tumorigenesis [72].

The majority of acquired AA and some RCC cases can be considered as T-cell-mediated autoimmune disease, resulting in bone marrow failure [74, 76]. Autoantibodies are detected in both conditions. However, their significance and pathophysiological significance in MDS and SAA is unclear [76].

Relative lymphocytosis, oligoclonal T-cell expansion, elevated cytokine levels are common features of AA and MDS, suggesting a common immune defect in the pathogenesis [72] of adults with low-grade MDS.

Decreased CD4 + FOXP3 + Treg cells, increased NK cells/impaired activity of NK cells, suppression of hematopoietic progenitors by cytotoxic CD8+ cells [72, 74], increased cytokines secreted by bone marrow microenvironment, macrophages (IF-, TNF-α which are proapoptotic), increased Th17 cells [interleukin ((IL)-17, IL-23, IL-1, and IL-6] which are cytotoxic to bone marrow precursors, decreased dendritic cells, decreased B cells [72], polyclonal hypo-, hypergammaglobulinemia, C3 hypocomplementemia, altered self-reactive antibody reper‐ toires [74] play role in the pathogenesis, some of which change as to the risk of MDS [72].

Some of these abnormalities overlap with those in autoimmune disorders themselves. The dysmorphic features in autoimmune disorders were delineated previously. Patients with autoimmune disorders should always be suspected for being MDS cases and should be evaluated for pathologic and genetic abnormalities.

#### **9. Common features in pathogenesis**

#### **9.1. Cytopenia**

Inherited bone marrow failure syndromes, MDS and SAA share the same pathogenetic features for involving a driver mutation, overproduction of cytokines and/or suppression of hematopoiesis through cytokines and deregulation of stem cell niche [76].

MDS and severe aplastic anemia share the same pathogenesis, as to abnormalities in T cells, especially in CD8+ cells; autoimmune manifestations and giving good response to immuno‐ suppressive therapy [76].

#### **9.2. Myelodysplasia in relation to cell cycle and other factors**

Myelodysplasia in blood cells arise due to any challenge during the course of normal differ‐ entiation in which cells exit the cell cycle and enter G0 phase permanently (**Figure 10a**)

Cell cycle control system depends on cyclically activated cyclin-dependent protein kinases (Cdks) a number of enzymes and other proteins, the most important being cyclins and other genes [118]. Stem cell differentiation is regulated by differentiation specific genes, homeotic genes, tumor suppressor genes, abnormality of which result in restriction in further prolifer‐ ation giving rise to alteration in normal cell cycle, and dysdifferentiation [119].

Hence, disturbance of expression of iron dependent genes regulating cell cycle in differentia‐ tion of hematopoietic cells in iron deficiency (see Section 2.5.2), deletion in human cell division cycle related gene (hCDCrel) in patients with del (22q11.2) are a few examples that lead to aforementioned myelodysplastic findings through genetic abnormalities. That the severity and spectrum of dysmorphic features in myelodysplasia differ according to the underlying secondary or primary pathology like in dysmegakaryopoiesis and thrombocytosis in inv 3(q21q26)/t(3;3)(q21;q26) and 5q-syndrome also reflect the various alterations playing role in different levels of differentiation.

Relative lymphocytosis, oligoclonal T-cell expansion, elevated cytokine levels are common features of AA and MDS, suggesting a common immune defect in the pathogenesis [72] of

Decreased CD4 + FOXP3 + Treg cells, increased NK cells/impaired activity of NK cells, suppression of hematopoietic progenitors by cytotoxic CD8+ cells [72, 74], increased cytokines secreted by bone marrow microenvironment, macrophages (IF-, TNF-α which are proapoptotic), increased Th17 cells [interleukin ((IL)-17, IL-23, IL-1, and IL-6] which are cytotoxic to bone marrow precursors, decreased dendritic cells, decreased B cells [72], polyclonal hypo-, hypergammaglobulinemia, C3 hypocomplementemia, altered self-reactive antibody reper‐ toires [74] play role in the pathogenesis, some of which change as to the risk of MDS [72].

Some of these abnormalities overlap with those in autoimmune disorders themselves. The dysmorphic features in autoimmune disorders were delineated previously. Patients with autoimmune disorders should always be suspected for being MDS cases and should be

Inherited bone marrow failure syndromes, MDS and SAA share the same pathogenetic features for involving a driver mutation, overproduction of cytokines and/or suppression of

MDS and severe aplastic anemia share the same pathogenesis, as to abnormalities in T cells, especially in CD8+ cells; autoimmune manifestations and giving good response to immuno‐

Myelodysplasia in blood cells arise due to any challenge during the course of normal differ‐ entiation in which cells exit the cell cycle and enter G0 phase permanently (**Figure 10a**)

Cell cycle control system depends on cyclically activated cyclin-dependent protein kinases (Cdks) a number of enzymes and other proteins, the most important being cyclins and other genes [118]. Stem cell differentiation is regulated by differentiation specific genes, homeotic genes, tumor suppressor genes, abnormality of which result in restriction in further prolifer‐

Hence, disturbance of expression of iron dependent genes regulating cell cycle in differentia‐ tion of hematopoietic cells in iron deficiency (see Section 2.5.2), deletion in human cell division cycle related gene (hCDCrel) in patients with del (22q11.2) are a few examples that lead to aforementioned myelodysplastic findings through genetic abnormalities. That the severity and spectrum of dysmorphic features in myelodysplasia differ according to the underlying secondary or primary pathology like in dysmegakaryopoiesis and thrombocytosis in inv

ation giving rise to alteration in normal cell cycle, and dysdifferentiation [119].

hematopoiesis through cytokines and deregulation of stem cell niche [76].

**9.2. Myelodysplasia in relation to cell cycle and other factors**

adults with low-grade MDS.

80 Myelodysplastic Syndromes

evaluated for pathologic and genetic abnormalities.

**9. Common features in pathogenesis**

**9.1. Cytopenia**

suppressive therapy [76].

We previously detected various temporary or permanent cell cycle abnormalities in total leukocytes, granulocytes and mononuclear cells from peripheral blood of ITP patients who had received steroids and a child with congenital neutropenia and her non-neutropenic mother all displaying myelodysplasia (**Figure 10**) [17, 45, 99].

The peripheral granulocytes (neutrophils and bands), monocytes, and lymphocytes are expected to be in G0 phase of the cell cycle, since they are terminally differentiated (**Fig‐ ure 10a**). We interpreted these numerous abnormal cell cycle configurations instead of G0 [99], as an alteration in differentiation, after a stimulus that was sensed as DNA damage, probably through disruption of one or more of these aforementioned enzymes [118]. Increased trilineage apoptosis, a type of cell death in some of these patients [45, 99], all having myelodysplasia are also in accordance of this interpretation.

Overlapping of a number of myelodysplastic features (**Tables 1**–**4**) closely with those of senescence-like phenotype (SLP) of rapid cell senescence (RCS), another type of cell death, led us to search RCS both in congenital neutropenia [45, 99] and autoimmune disorders (JRA, SLE, and ITP) all having myelodysplasia [17, 28, 30, 31, 79]. We detected that three children with congenital neutropenia and their non-neutropenic mothers [45, 99] and all patients with autoimmune disorders displayed RCS in their leukocytes shown by β-galactosidase (SA-β-gal) positivity [79] (**Figure 1**). In several patients, cell cycle abnormalities accompanied [99] (**Figure 10I**).

The RCS that was detected in autoimmune disorders and those with congenital neutropenia [45] were attributed to increased proinflammatory cytokines and chemokines in autoimmune disorders [79] and congenital neutropenia [45] through giving rise to loss of telomeres by keeping the immune system in a state of low level of activation [79]. Absence of RCS (unpub‐ lished data) in iron deficient patients was thought to be due to absence of increase in proin‐ flammatory cytokines, confirming this hypothesis.

Differentiation is controlled not only by intracellular genetic factors but by extracellular factors like extracellular matrix and soluble factors like fibroblast growth factor (FGF), transforming growth factor beta (TGFB), colony stimulating factor-1 (CSF-1), GCSF, GM-CSF, stem cell factor (SCF), Fms-like tyrosine kinase 3 ligand (Flt-3 ligand), ILs as well [119].

The reasons that we previously discussed in secondary myelodysplasia like immune destruc‐ tion of stromal and hematopoietic cells, inhibitory effects of cytokines and/or other intracel‐ lular/extracellular messengers/soluble factors, on the microenvironment and/or hematopoietic cells; decreased differentiation, regeneration and increased clearance of stem cells, reflected by alterations in cell cycle, impairment of heme synthesis and iron utilization also play role in dysdifferentiation.

Therefore, myelodysplasia either primary or secondary is associated with cell death parame‐ ters, and cell cycle alterations. It reflects the viability of the cell and can be assumed as a tip of a big iceberg that is an harbinger of a large spectrum of primary (clonal) and secondary (nonclonal) disorders. We think that the criteria of dysplasia should be revised in the definition of MDS and attention should be exercised to find out practical laboratory means to detect clonality.

#### **10. Differential diagnosis**

Discrimination between low-risk MDS and disorders mimicking MDS depends on determin‐ ing whether hematopoiesis is clonal or not.

To assess clonality and dysplasia, flow cytometric evaluation is promising [58, 120, 121]. The minimal requirements to assess dysplasia by flow cytometry have been defined for adulthood low-risk MDS as in the following [120]:

(a) For immature myeloid and monocytic progenitors: Increased percentage of cells in nucleated cell fraction, lack of/decreased/increased expression of CD45, CD34, CD13 + CD33, homogenous under/overexpression of CD117, lack of/increased expression of HLA-DR, asynchronous expression of CD11b, CD15, expression of CD5, CD7, CD19, CD56 which are lineage infidelity markers.

(b) For maturing neutrophils: Decreased percentage of cells as ratio to lymphocytes, sideward light scatter (SSC) as ratio vs SSC of lymphocytes, altered pattern in relationship of CD13 with CD11b and CD13 with CD16, CD15 with CD10 (like lack of CD10 on mature neutrophils).

(c) For monocytes: Decreased or increased percentage of cells, shift toward immature distri‐ bution, altered pattern in relationship of HLA-DR with CD11b and CD36 with CD14, homo‐ genous under or overexpression of CD13 and CD33, expression of CD56 as which is a lineage infidelity marker.

(d) Progenitor B cells: Decreased or absent progenitor B cells when enumeration is performed as fraction of total CD34+ based on CD45/CD34/SSC in combination with CD10 or CD19.

(e) Erythroid compartment: Increased percentage of nucleated erythroid cells, altered pattern in relationship of CD71 with CD235a, decreased expression of CD71 and CD36, increased percentage of CD117-positive precursors.

(f) For megakaryocytes: No standard application of flow cytometry has been described for megakaryocytes yet.

World Health Organization recognized more than three flow cytometric aberrancies as indicative of MDS [120]. It was also reported that two or more aberrancies in only the four parameters as increased percentage of CD34+ progenitor cells in bone marrow, decreased number of progenitor B cells within the CD34+ compartment, decreased or increased CD45 expression on myeloid progenitor cells and decreased SSC of neutrophils, CD10, CD15, CD11b, CD56 being additional useful markers could identify 70% of low-risk MDS cases with 94% specificity [120]. In children, for distinction between SAA and RCC, which generally present with hypocellular bone marrow, a cutoff of 2 flow cytometric abnormalities was found to have 60% sensitivity and 88% specificity which changed as 76% and 84% respectively, when combined with other diagnostic parameters [121]. In nonclonal disorders either no or only one flow cytometric abnormality was found. In low-risk MDS, in rare patients no flow-cytometric abnormality was found [58].

However, abnormal flow patterns were encountered in AML, MPN, and natural aging also [58]. Current knowledge on normal and abnormal patterns in the elderly and in normal controls is still inadequate [120].

The flow cytometric analysis of children with RCC (low-risk MDS in childhood) showed no difference in the relative SSC of granulocytes between those in RCC and healthy controls and absence of lineage infidelity markers on myeloid blasts unlike commonly occurring in adult low-risk MDS. The most frequent abnormality in RCC was reported to be heterogeneous expression of CD71 and CD36 on erythrocytes and aberrant expression of CD56 on monocytes (in 58 and 20%). All other abnormalities were observed in RCC in lower frequency than in adulthood low-risk MDS [121].

However, although flow cytometric evaluation is promising in diagnosis of MDS cases which lacked specific diagnostic markers like ring sideroblasts or karyotypic aberrations, it can only be used as a part of a diagnostic work-up consisting of histopathology and cytogenetic analysis [120, 121].

Flow cytometry can be used to rule out PNH; but minor PNH clones are present in 13–23% of adult MDS, and 41% of children with RCC [121].

#### **11. Future recommendations**

(nonclonal) disorders. We think that the criteria of dysplasia should be revised in the definition of MDS and attention should be exercised to find out practical laboratory means to detect

Discrimination between low-risk MDS and disorders mimicking MDS depends on determin‐

To assess clonality and dysplasia, flow cytometric evaluation is promising [58, 120, 121]. The minimal requirements to assess dysplasia by flow cytometry have been defined for adulthood

(a) For immature myeloid and monocytic progenitors: Increased percentage of cells in nucleated cell fraction, lack of/decreased/increased expression of CD45, CD34, CD13 + CD33, homogenous under/overexpression of CD117, lack of/increased expression of HLA-DR, asynchronous expression of CD11b, CD15, expression of CD5, CD7, CD19, CD56 which are

(b) For maturing neutrophils: Decreased percentage of cells as ratio to lymphocytes, sideward light scatter (SSC) as ratio vs SSC of lymphocytes, altered pattern in relationship of CD13 with CD11b and CD13 with CD16, CD15 with CD10 (like lack of CD10 on mature neutrophils).

(c) For monocytes: Decreased or increased percentage of cells, shift toward immature distri‐ bution, altered pattern in relationship of HLA-DR with CD11b and CD36 with CD14, homo‐ genous under or overexpression of CD13 and CD33, expression of CD56 as which is a lineage

(d) Progenitor B cells: Decreased or absent progenitor B cells when enumeration is performed as fraction of total CD34+ based on CD45/CD34/SSC in combination with CD10 or CD19.

(e) Erythroid compartment: Increased percentage of nucleated erythroid cells, altered pattern in relationship of CD71 with CD235a, decreased expression of CD71 and CD36, increased

(f) For megakaryocytes: No standard application of flow cytometry has been described for

World Health Organization recognized more than three flow cytometric aberrancies as indicative of MDS [120]. It was also reported that two or more aberrancies in only the four parameters as increased percentage of CD34+ progenitor cells in bone marrow, decreased number of progenitor B cells within the CD34+ compartment, decreased or increased CD45 expression on myeloid progenitor cells and decreased SSC of neutrophils, CD10, CD15, CD11b, CD56 being additional useful markers could identify 70% of low-risk MDS cases with 94% specificity [120]. In children, for distinction between SAA and RCC, which generally present with hypocellular bone marrow, a cutoff of 2 flow cytometric abnormalities was found to have

clonality.

82 Myelodysplastic Syndromes

**10. Differential diagnosis**

ing whether hematopoiesis is clonal or not.

low-risk MDS as in the following [120]:

percentage of CD117-positive precursors.

lineage infidelity markers.

infidelity marker.

megakaryocytes yet.

We recommend that all the aforementioned disorders be considered in the differential diagnosis of MDS. Patients who do not comply with none of a definite diagnosis should be followed-up for a considerable time period in order to assure a spontaneous remission or progression. Spontaneous remission in children and youngsters were reported as 2.2–30 months after the diagnosis [61–68]. In childhood, in the setting of hypocellular bone marrow with absence of cytogenetic abnormality [4] or a bone marrow biopsy with topography and cellularity of the local hematopoiesis [9], two bone marrow biopsies at least two weeks apart are necessary [4]. For cases with refractory cytopenia and cases with less than 15% ring sideroblasts all of which display unilineage dysplasia without excess blasts, repeated bone marrow examination is recommended after a 6 months' observation [5].

Since morphologic dysplasia can be encountered in both clonal and nonclonal disorders, the criteria of dysplasia should be revised in the definition of MDS and attention should be exercised to find out practical laboratory means to detect clonality. Flow cytometry is a promising means to distinguish between clonal and nonclonal cytopenia when used together with other diagnostic tools.

#### **Acknowledgements**

The authors thank Hamza Okur PhD, for kindly performing flow cytometric analysis of cell cycle, Prof. Dr Esra Erdemli and Deniz Billur MD, for analyzing the leukocytes for rapid senescence.

#### **Author details**

Lale Olcay1\* and Sevgi Yetgin2

\*Address all correspondence to: baskent.edu.tr

1 Department of Pediatrics, Başkent University Faculty of Medicine, Unit of Pediatric Hematology, Oncology, Ankara, Turkey

2 Department of Pediatrics, Hacettepe University Faculty of Medicine, Unit of Pediatric Hematology, Oncology, Ankara, Turkey

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84 Myelodysplastic Syndromes

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2 Department of Pediatrics, Hacettepe University Faculty of Medicine, Unit of Pediatric

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## **Myelodysplastic Disorders, 5q-Syndrome**

#### Khalid Ahmed Al-Anazi

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64202

**Abstract**

The myelodysplastic syndromes (MDSs) are characterized by ineffective erythropoie‐ sis and progressive cytopenia and ultimately affected patients develop acute myeloid leukemia (AML) or die from advanced bone marrow (BM) failure.

Myelodysplastic syndrome (MDS) with isolated del (5q) is a common type of MDS with specific pathological and clinical manifestations including refractory anemia. It is usually treated by (1) supportive measures including blood transfusions that may cause iron overload that requires iron chelation therapy, (2) targeted therapies such as the immunomodulatory drug lenalidomide, and (3) hematopoietic stem cell transplanta‐ tion (HSCT) in transplant eligible individuals. The establishment of the various prognostic systems, the discovery of the new genetic mutations, and the identification of new targets, in MDSs in general and in 5q-syndrome in particular, will hopefully translate into more pinpointed targeted therapies that will further improve the outcomes of patients having these disorders.

**Keywords:** myelodysplastic syndrome, 5q-syndrome, iron overload, lenalidomide, hematopoietic stem cell transplantation

#### **1. Introduction**

The MDSs are a group of clonal stem cell disorders that are characterized by ineffective erythropoiesis due to excessive apoptosis and progressive peripheral blood cytopenia culminating into acute myeloid leukemia AML or death from progressive BM failure [1–4]. MDS is primarily a disease of the elderly with a median age of 70 years [3]. The MDSs have been linked to several etiologies, risk factors, and environmental associations such as alcohol intake, tobacco use, Sweet's syndrome, vitamin deficiencies, cytotoxic chemotherapy, various hereditary disorders, and BM failure syndromes [5–12].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2. Pathogenesis of MDSs**

The pathogenesis of MDS is poorly understood [5, 12]. However, several pathogenic mecha‐ nisms have been described and these include the following: (1) genetic mutations as the cell of origin has acquired multiple mutations that result in dysplasia and ineffective erythropoi‐ esis; (2) MDS clonality: MDS is a clonal process thought to develop from a single-transformed hematopoietic progenitor cell. The inciting mutation is unknown for the majority of cases. However, recurrent genetic mutations involving RNA splicing machinery have been identi‐ fied; (3) haploinsufficiency of ribosomal proteins particularly ribosomal protein (RPS) 14 in del (5q); (4) telomere dysfunction and aberrant or absent expression of micro-RNA species; (5) epigenetic changes: MDS genomes are characterized by global DNA hypomethylation with concomitant hypermethylation of gene-promoter regions relative to normal controls; (6) factors extrinsic to hematopoietic cells such as stromal abnormalities and T-cell dysregulation that may occur causally or secondary to the primary genetic defects; (7) accelerated apoptosis and ineffective erythropoiesis; (8) altered immune responses such as polyclonal expansion of helper T cells (CD4+) and oligoclonal expansion of cytotoxic T cells (CD8+) in the peripheral blood and BM; (10) leukemic transformation in MDS; the estimated risk of leukemic transfor‐ mation is more than 50% and is more frequent in patients with high-risk MDS such as refractory anemia with excess of blasts (RAEB) II, monosomy 7, deletion of short arm of chromosome 17, deletion of long arm of chromosome 7, and trisomy 8 [5, 12].

#### **3. Chromosome 5 abnormalities in MDS**

Approximately, 15% of patients with MDS have abnormalities of chromosome 5 that include insertional deletion of a segment of the long arm of the chromosome [del (5q) or 5q-syndrome], monosomy 5, and unbalanced translocations [13].

#### **3.1. Del (5q) type of MDS**

The insertional deletion of the long arm of chromosome 5, del (5q), is the one of the most common cytogenetic abnormality encountered in patients with MDSs as it has been reported in 10–30% of patients with MDS [14–17]. The long arm of chromosome 5 has two distinct commonly deleted regions (CDRs). The more distal CDR lies in 5q33.1 and contains 40 protein coding genes and genes that code for microRNAs (miR-143 and miR-145) [13]. Many genes related to hematopoiesis are located on the long arm of chromosome 5 [18]. In del (5q), one allele is deleted and this accounts for the genetic haploinsufficiency [13]. The gene cluster at 5q31 includes interleukins (ILs) 3, 4, 5, 9, 13, and 17β in addition to granulocyte monocytecolony-stimulating factor (CSF). Several cytokine receptor genes are also located on the long arm of chromosome 5 including: CSF-1 receptor and platelet-derived growth factor-β [18].

The world health organization (WHO) recognizes del (5q), which was first described by Van den Berghe et al. in the year 1974, as a distinct form of MDS [15, 18–20]. The 5q-syndrome is the most distinct type of all MDSs as it has clear genotype/phenotype relationship [18, 21]. If del (5q) occurs as the only cytogenetic abnormality, it is associated with favorable prognosis but once it is encountered in association with other single or multiple chromosomal abnor‐ malities, particularly in the setting of complex cytogenetics, the clinical outcome is rendered poor [14, 16, 20, 22, 23].

**2. Pathogenesis of MDSs**

96 Myelodysplastic Syndromes

deletion of long arm of chromosome 7, and trisomy 8 [5, 12].

**3. Chromosome 5 abnormalities in MDS**

monosomy 5, and unbalanced translocations [13].

**3.1. Del (5q) type of MDS**

The pathogenesis of MDS is poorly understood [5, 12]. However, several pathogenic mecha‐ nisms have been described and these include the following: (1) genetic mutations as the cell of origin has acquired multiple mutations that result in dysplasia and ineffective erythropoi‐ esis; (2) MDS clonality: MDS is a clonal process thought to develop from a single-transformed hematopoietic progenitor cell. The inciting mutation is unknown for the majority of cases. However, recurrent genetic mutations involving RNA splicing machinery have been identi‐ fied; (3) haploinsufficiency of ribosomal proteins particularly ribosomal protein (RPS) 14 in del (5q); (4) telomere dysfunction and aberrant or absent expression of micro-RNA species; (5) epigenetic changes: MDS genomes are characterized by global DNA hypomethylation with concomitant hypermethylation of gene-promoter regions relative to normal controls; (6) factors extrinsic to hematopoietic cells such as stromal abnormalities and T-cell dysregulation that may occur causally or secondary to the primary genetic defects; (7) accelerated apoptosis and ineffective erythropoiesis; (8) altered immune responses such as polyclonal expansion of helper T cells (CD4+) and oligoclonal expansion of cytotoxic T cells (CD8+) in the peripheral blood and BM; (10) leukemic transformation in MDS; the estimated risk of leukemic transfor‐ mation is more than 50% and is more frequent in patients with high-risk MDS such as refractory anemia with excess of blasts (RAEB) II, monosomy 7, deletion of short arm of chromosome 17,

Approximately, 15% of patients with MDS have abnormalities of chromosome 5 that include insertional deletion of a segment of the long arm of the chromosome [del (5q) or 5q-syndrome],

The insertional deletion of the long arm of chromosome 5, del (5q), is the one of the most common cytogenetic abnormality encountered in patients with MDSs as it has been reported in 10–30% of patients with MDS [14–17]. The long arm of chromosome 5 has two distinct commonly deleted regions (CDRs). The more distal CDR lies in 5q33.1 and contains 40 protein coding genes and genes that code for microRNAs (miR-143 and miR-145) [13]. Many genes related to hematopoiesis are located on the long arm of chromosome 5 [18]. In del (5q), one allele is deleted and this accounts for the genetic haploinsufficiency [13]. The gene cluster at 5q31 includes interleukins (ILs) 3, 4, 5, 9, 13, and 17β in addition to granulocyte monocytecolony-stimulating factor (CSF). Several cytokine receptor genes are also located on the long arm of chromosome 5 including: CSF-1 receptor and platelet-derived growth factor-β [18]. The world health organization (WHO) recognizes del (5q), which was first described by Van den Berghe et al. in the year 1974, as a distinct form of MDS [15, 18–20]. The 5q-syndrome is the most distinct type of all MDSs as it has clear genotype/phenotype relationship [18, 21]. If

Patients with del (5q) have specific clinical and pathological features [15, 18, 20]. The 5 qsyndrome is usually characterized by the following: (1) female predominance, (2) refractory macrocytic anemia that is often severe, (3) normal or elevated platelet count, (4) BM findings of erythroid hypoplasia, less than 5% blasts as well as abnormal, dysplastic or hypolobulated megakaryocytes, (5) del (5q) chromosomal abnormality as the sole karyotypic abnormality, and (6) a rather benign clinical course with approximately 10% of patients ultimately pro‐ gressing to AML [13, 14, 18–20].

Despite the remarkable progress that has been achieved recently, certain unclear issues related to the pathogenesis of del (5q) need further evaluation [18, 20]. Del (5q) MDS is considered a disorder of the hematopoietic stem cells with lympho-myeloid potential. Also, involvement of B cells, rather than T cells, was documented by combining immunophenotyping and fluorescence *in situ* hybridization (FISH) analysis [18]. Cytogenetic and FISH analysis in BM progenitor cells have revealed that, in del (5q) MDS, the deletion was generally present in the pluripotent hematopoietic stem cells (CD34+ and CD38+) with the persistence of the normal progenitor cells in the BM [20]. Genomic stability in 5q-syndrome is related to the infrequency of additional cytogenetic abnormalities [18]. The lack of mutations in the genes mapping the CDR suggests that haploisufficiency is the basis of 5q-syndrome [13, 18, 20]. Candidate genes that show haploinsufficiency in del (5q) include SPARC (secreted protein acidic and cysteine rich), a tumor suppressor gene, and RPS14, which is a component of the 40s ribosomal subunit [13]. Only in advanced forms of the disease, rare mutations involving p53, JAK2, and MPL genes have been described [13, 18, 20].

The erythroid defect or failure in del (5q) appears to be multifactorial as it has been reported to involve in the following: (1) the decreased expression or haploinsufficiency of the ribosomal protein S14 [RPS14] gene, (2) the upregulation of the p53 pathway induced by ribosomal stress, and (3) enhancement of the endogenous erythropoietin production that ultimately leads to red cell transfusion dependence in most patients [13, 15, 18, 21]. On the other hand, loss of the microRNA genes miR-145 and miR-146a has been associated with the thrombocytosis observed in 5q-syndrome patients [21]. Also, the increased expression of Friend leukemia virus integration 1(FLI1), which is one of the target genes of miR-145, maintains effective megakar‐ yopoiesis in del (5q) MDS resulting in normal or elevated platelet (PLT) counts [13].

Isolated del (5q) has been reported in in higher grade MDSs such as RAEB and RAEBthrombocytosis (RAEB-T), thus contributing to the heterogeneity of the disease [20, 22]. MDS with del (5q) occurs not only in myelodysplastic disorders, but also in AML and it contributes to the pathogenesis of both myeloid diseases by deleting one or more of the tumor suppressor genes [22]. Once associated with additional cytogenetic abnormalities and once new genetic mutations are acquired, such as TP53 mutation, MDS with del (5q) becomes an aggressive disease with rapid evolution into AML [20, 22]. Therefore, isolated del (5q) MDS should be differentiated from other forms of myelodysplasia having del (5q) associated with other cytogenetic abnormalities and an excess of BM blasts [20]. Other specific aspects of 5qsyndrome will be discussed separately in the subsequent sections of the review manuscript.

#### **3.2. Monosomy 5 type of MDS**

Loss of the whole chromosome 5 has been described in about 3–8% MDS cases. Recent studies have shown that many suspected monosomies 5 are in fact cryptic translocations or insertions, undetectable by conventional G-banding [24]. The mechanism responsible for the fragmenta‐ tion of deleted chromosome 5 remains unclear. One of the possible explanations might be the phenomenon called chromothripsis, whereby one or more chromosomes or chromosomal regions shatter into pieces in a single catastrophic event. MDS patients with deleted chromo‐ some 5 involved in complex rearrangements should be considered as a unique entity with extremely poor prognosis [24].

Monosomy 5 does exist but is rarely encountered and the presence of this chromosomal abnormality is usually associated with complex karyotypes, conferring poor prognosis [25]. Studies have shown that, compared to 5q- syndrome, monosomy 5 is more frequently associated with: advanced or higher risk MDS, other chromosomal abnormalities including chromosome 7 abnormalities and inferior overall survival [26]. Monosomy 5 has been reported in therapy-related MDS (t-MDS) with complex cytogenetics and rapid progression to death [27].

#### **4. Clinical manifestations and complications of MDS**

MDS has nonspecific signs and symptoms at presentation. However, many patients are asymptomatic at presentation. The main manifestations of MDS are those related to cytopenia. Anemic manifestations include fatigue, weakness, dizziness, exercise intolerance, angina, cognitive impairment, and altered sense of well-being [12, 28]. Patients having thrombocyto‐ penia present with bleeding from various sites such as skin and mucous membranes. Easy bruising, epistaxis, petechiae, ecchymoses, and gum bleeding are the main manifestations [12, 28]. Patients with neutropenia may develop fever and infections may be due to viruses, bacteria, fungi, and mycobacteria [12, 28, 29]. Physical examination in patients with MDS usually reveals: pallor, petechiae or ecchymoses, hepatosplenomegaly, and lymphadenopathy uncommonly, weight loss in advanced cases and skin manifestations in case of associated Sweet's syndrome [12, 28].

Autoimmune abnormalities may be present in MDS patients and they include cutaneous vasculitis, monoarticular arthritis, pericarditis, pleural effusions, edema formation, skin ulcerations, iritis, myositis, peripheral neuropathy, fever, and pulmonary infiltrates [12]. Other abnormalities that may be encountered in patients with MDS include pure red cell aplasia, acquired HbH disease, myeloid sarcomas, Sweet's syndrome, myocardial ischemia, and thrombocytosis in patients with del (5q) and RARS-T [12, 28].

#### **5. Diagnosis and subtypes of MDS**

cytogenetic abnormalities and an excess of BM blasts [20]. Other specific aspects of 5qsyndrome will be discussed separately in the subsequent sections of the review manuscript.

Loss of the whole chromosome 5 has been described in about 3–8% MDS cases. Recent studies have shown that many suspected monosomies 5 are in fact cryptic translocations or insertions, undetectable by conventional G-banding [24]. The mechanism responsible for the fragmenta‐ tion of deleted chromosome 5 remains unclear. One of the possible explanations might be the phenomenon called chromothripsis, whereby one or more chromosomes or chromosomal regions shatter into pieces in a single catastrophic event. MDS patients with deleted chromo‐ some 5 involved in complex rearrangements should be considered as a unique entity with

Monosomy 5 does exist but is rarely encountered and the presence of this chromosomal abnormality is usually associated with complex karyotypes, conferring poor prognosis [25]. Studies have shown that, compared to 5q- syndrome, monosomy 5 is more frequently associated with: advanced or higher risk MDS, other chromosomal abnormalities including chromosome 7 abnormalities and inferior overall survival [26]. Monosomy 5 has been reported in therapy-related MDS (t-MDS) with complex cytogenetics and rapid progression to death

MDS has nonspecific signs and symptoms at presentation. However, many patients are asymptomatic at presentation. The main manifestations of MDS are those related to cytopenia. Anemic manifestations include fatigue, weakness, dizziness, exercise intolerance, angina, cognitive impairment, and altered sense of well-being [12, 28]. Patients having thrombocyto‐ penia present with bleeding from various sites such as skin and mucous membranes. Easy bruising, epistaxis, petechiae, ecchymoses, and gum bleeding are the main manifestations [12, 28]. Patients with neutropenia may develop fever and infections may be due to viruses, bacteria, fungi, and mycobacteria [12, 28, 29]. Physical examination in patients with MDS usually reveals: pallor, petechiae or ecchymoses, hepatosplenomegaly, and lymphadenopathy uncommonly, weight loss in advanced cases and skin manifestations in case of associated

Autoimmune abnormalities may be present in MDS patients and they include cutaneous vasculitis, monoarticular arthritis, pericarditis, pleural effusions, edema formation, skin ulcerations, iritis, myositis, peripheral neuropathy, fever, and pulmonary infiltrates [12]. Other abnormalities that may be encountered in patients with MDS include pure red cell aplasia, acquired HbH disease, myeloid sarcomas, Sweet's syndrome, myocardial ischemia, and

**4. Clinical manifestations and complications of MDS**

thrombocytosis in patients with del (5q) and RARS-T [12, 28].

**3.2. Monosomy 5 type of MDS**

98 Myelodysplastic Syndromes

extremely poor prognosis [24].

Sweet's syndrome [12, 28].

[27].

Minimal morphological diagnostic criteria in MDS include the following: (1) BM findings: ≥ 10% dysplastic cells in ≥ 1 myeloid lineages, (2) highly suggestive features: (a) granulocytic series: agranular neutrophils and Pelger–Huet neutrophils, (b) megakaryocytes, small binucleated megakaryocytes and small round separated nuclei in megakaryocytes, and (c) erythroid series: multinuclear or asymmetrical nuclei, nuclear bridging, and ring sideroblasts [6, 30, 31].

The minimal diagnostic criteria in MDS include the following: (1) prerequisite criteria that include (a) constant cytopenia in ≥ 1 of the following lineages: erythroid: Hb < 11 g/dL, neutrophilic; absolute neutrophil count (ANC) < 1.5 × 109 /L or megakaryocytic, PLTs < 100 × 109 /L. (b) exclusion of all other hematopoietic and nonhematopoietic disorders as primary reasons for cytopenia or dysplasia; (2) MDS-related or decisive criteria: (a) dysplasia in at least 10% of all cells in one of the following lineages in the BM smear; erythroid, neutrophilic or megakaryocytic or > 15% ring sideroblasts on iron staining, (b) 5–19% blasts on BM smears, and (c) typical chromosomal abnormality by FISH or conventional karyotype. (3) cocriteria for patients fulfilling (1) but not (2): (a) abnormal phenotype of BM cells clearly indicative of a monoclonal population of erythroid and/ or myeloid cells determined by flow cytometry, (b) clear molecular signs of a monoclonal cell population in human androgen receptor (HU‐ MARA) assay, gene chip profiling or point mutation analysis such as RAS mutation, (c) markedly or persistently reduced colony formation of BM and/or circulating progenitor cells by colony-forming unit assay [6, 30, 31]. The subtypes of MDS according to the WHO classi‐ fication are illustrated in **Table 1** [6, 30, 31].



MDS: myelodysplastic syndrome; PLT: platelet; ↑: increased.

**Table 1.** WHO classification of MDS.

#### **5.1. Cytogenetics in MDS**

Several cytogenetic abnormalities can be encountered in patients with MDSs, some of these are balanced, while others are unbalanced as illustrated in **Table 2** [6]. Cytogenetic abnormal‐ ities are major determinants in the pathogenesis of MDS. They are becoming increasingly recognized as the basis of selecting drugs in individual patients with MDS and they play a significant role in monitoring response to treatment [32]. Chromosomal abnormalities are detected in approximately 50% of patients with *de novo* MDS and 80% of patients with t-MDS [32]. Recently, our ability to define the prognosis of the individual patient with MDS has improved significantly [6]. Cytogenetic abnormalities are becoming essential in determining the prognosis of MDS because they constitute the basis of the new cytogenetic scoring system as shown in **Table 3** [31, 33, 34]. The values of the new prognostic systems will certainly become higher as new genetic-based therapy move through trials and into clinical practice [6].


\*MDS: myelodysplastic syndrome.

**Subtype of MDS Proportion of MDS**

**RARS**

**RCMD**

**MDS-U**

[unclassified MDS]

sideroblasts]

[refractory anemia ring

100 Myelodysplastic Syndromes

[refractory anemia with multi lineage dysplasia with or without ring sideroblasts]

**patients**

MDS with isolated del (5q) Uncommon Anemia

MDS: myelodysplastic syndrome; PLT: platelet; ↑: increased.

**Table 1.** WHO classification of MDS.

**5.1. Cytogenetics in MDS**

Unknown percentage

or bicytopenia] ↓ PLT: < 1% No or < 1% blasts sideroblasts

3–11% Anemia

30% Cytopenia(s)

**Peripheral blood findings**

No blasts

< 1% blasts No Auer rods Monocytes < 1 G/L

Normal or high PLT count < 1% blasts

Cytopenia ≤ 1% blasts

Several cytogenetic abnormalities can be encountered in patients with MDSs, some of these are balanced, while others are unbalanced as illustrated in **Table 2** [6]. Cytogenetic abnormal‐ ities are major determinants in the pathogenesis of MDS. They are becoming increasingly recognized as the basis of selecting drugs in individual patients with MDS and they play a significant role in monitoring response to treatment [32]. Chromosomal abnormalities are detected in approximately 50% of patients with *de novo* MDS and 80% of patients with t-MDS [32]. Recently, our ability to define the prognosis of the individual patient with MDS has improved significantly [6]. Cytogenetic abnormalities are becoming essential in determining the prognosis of MDS because they constitute the basis of the new cytogenetic scoring system as shown in **Table 3** [31, 33, 34]. The values of the new prognostic systems will certainly become higher as new genetic-based therapy move through trials and into clinical practice [6].

**Bone marrow findings**

Unilineage erythroid

≥ 15% ring siderblasts

± 15% ring sideroblasts

Dysplasia in < 10% cells but cytogenetic

abnormalities are considered

5% blasts without Auer rods Megakaryocytes: normal or ↑;

belonging to at least 2 cell lines < 5% blasts without

Auer rods

hypolobulated

presumptive for

MDS < 5% blasts

Dysplasia in ≥ 10% of cells

dysplasia < 5% blasts

**Table 2.** Chromosomal abnormalities in MDS.


MDS: myelodysplastic syndrome; AML: acute myeloid leukemia.

**Table 3.** New cytogenetic scoring system for MDS.

#### **5.2. Impact of monosomal karyotype on the prognosis of MDS**

A monosomal karyotype (MK) is defined by the presence of ≥ 2 distinct autosomal chromosome monosomies or a single autosomal monosomy associated with ≥ 1 structural abnormality [1]. In AML, MK has been associated with a worse prognosis than an otherwise complex karyo‐ type, regardless the specific type of autosome involved [1].

Studies have shown that MK in MDS identifies a prognostically worse subgroup of patients than a complex karyotype regardless of whether monosomy 7 or 5 is part of the MK component [1]. Chromosomal abnormalities are present in 20–70% of patients with MDS, but complex cytogenetics are universally considered unfavorable as they are associated with poor overall survival (OS) and high rates of leukemic transformation [1, 32, 34].

#### **5.3. Genetic mutations described in MDSs and 5q-syndrome**

Several classes of genetic mutations have been described in patients with MDS as shown in **Tables 4**–**6** [34–40]. These mutations are essential in not only determining the prognosis but also constituting a platform for the current and future novel and targeted therapies for various types of myelodysplasia (**Tables 4** and **6**) [34–39].



**5.2. Impact of monosomal karyotype on the prognosis of MDS**

type, regardless the specific type of autosome involved [1].

survival (OS) and high rates of leukemic transformation [1, 32, 34].

**location**

**5.3. Genetic mutations described in MDSs and 5q-syndrome**

types of myelodysplasia (**Tables 4** and **6**) [34–39].

**Class Mutation Chromosomal**

[Splicing factor 3b, subunit 1]

[Serine/arginine-rich splicing factor-2]

[U2 small nuclear RNA auxiliary factor-1]

[Zinc finger RNAbinding mofit and serine/arginine rich 2]

**SF3B1**

**SRSF2**

**U2AF1**

**ZRSR2**

**(1) RNA- splicing machinery (50%)**

102 Myelodysplastic Syndromes

A monosomal karyotype (MK) is defined by the presence of ≥ 2 distinct autosomal chromosome monosomies or a single autosomal monosomy associated with ≥ 1 structural abnormality [1]. In AML, MK has been associated with a worse prognosis than an otherwise complex karyo‐

Studies have shown that MK in MDS identifies a prognostically worse subgroup of patients than a complex karyotype regardless of whether monosomy 7 or 5 is part of the MK component [1]. Chromosomal abnormalities are present in 20–70% of patients with MDS, but complex cytogenetics are universally considered unfavorable as they are associated with poor overall

Several classes of genetic mutations have been described in patients with MDS as shown in **Tables 4**–**6** [34–40]. These mutations are essential in not only determining the prognosis but also constituting a platform for the current and future novel and targeted therapies for various

**Frequency Prognostic**

2q33.1 15–60% Good **– Phenotype:** ring

17q22.3 6–20% Poor **– MDS types:**

21q22.3 5–12% Unclear/poor **– MDS types:**

Xp22.1 3–10% Unknown **– MDS types:**

**significance**

**– Associations: phenotypes and MDS types – Application to treatment**

sideroblasts

– RCMD – RAEB – CMML

– RCMD – RAEB – CMML

– RCMD – RAEB – CMML

**– MDS types:** – RARS – RCMD – RS – RARS – T



**Class Mutation Chromosomal**

**(kinase signaling)** RAS viral oncogene

104 Myelodysplastic Syndromes

homolog]

**CBL** [cbl proto-

**JAK 2**

**FLT3** [Fms-related tyrosine -kinase 3]

**(4)**

**Cohesin familycomplex pathway** [Janus kinase 2]

oncogene E3 ubiquitin protein ligase]

**location**

**KRAS** 12 p12.1 2–6% Unclear/

**Frequency Prognostic**

**significance**

– Increased risk of progression to AML

adverse – Increased risk of progression to AML

– Does not appear to alter prognosis

prognosis – Progression to AML

prognosis

prognosis

prognosis

prognosis

<1% – Adverse

11q 23.3 1–5% Unknown – **MDS types:**

9p24 6.2–8.3% Unknown

**NF1** – <5% Poor – **MDS types:**

13 q12 <5% – Poor

**RAD 21** 8 p24 2% – Adverse

**STAG 2** X q25 5–10% – Adverse

**SMC 3** 10q25 2% – Adverse

P11.121

**SMC1 A** Xp 11.22-

**– Associations: phenotypes and MDS types – Application to treatment**

– CMML – JMML

– **MDS types**: – All MDS types – CMML – JMML

– All MDS types – CMML – JMML

– **Phenotype:** – Megakaryocytosis – **MDS types:** – all types/RARS-T/RA – JAK2 Inhibitors

– all MDS types – JMML

– **MDS types:** – All MDS types – FLT 3 Inhibitors

– **MDS types:** – RCMD – CMML – RAEB

–

–

–


MDS: myelodysplastic syndrome; CMML: chronic myelomonocytic leukemia; JMML: juvenile myelomonocytic leukemia; AML: acute myeloid leukemia; RCMD: refractory cytopenia with multilineage dysplasia; RA: refractory anemia; RAEB: refractory anemia with excess of blasts; RARS: refractory anemia with ring sideroblasts; RARS-T: refractory anemia with ring sideroblasts thrombocytosis.

**Table 4.** Genetic mutations in MDS.

TP53 encodes a cytoplasmic protein p53 that regulates cell growth and death. TP53 mutations have been found mainly in intermediate to high-risk MDS patients [41]. Patients having TP53 mutations often present with severe thrombocytopenia, complex cytogenetic abnormalities, an increased risk of leukemic transformation, and a shorter survival [41, 42]. Patients with mutant p53, compared to patients carrying wild-type p53, have the following features: older age, anemia, and leucopenia at the time of diagnosis and shorter median survival. Molecular identification of mutant p53 contributes to the risk stratification of patients with lower-risk MDS that may alter the treatment approach [41]. TP53 mutations develop at an early disease stage in almost 20% of patients with lower-risk MDS having del (5q) [42].


**Table 5.** Genetic mutations associated with poor prognosis in MDS.

**Class Mutation Chromosomal**

[V-KitHardy-Zuckerman 4 Feline sarcoma viral oncogene homolog]

[GNAS complex 10ms]

refractory anemia with ring sideroblasts thrombocytosis.

**Table 4.** Genetic mutations in MDS.

**GNAS**

**PTPN11** Protein tyrosine phosphatase nonreceptor type11

**(6) Other genetic mutations**

106 Myelodysplastic Syndromes

**location**

**PTEN** 10q 23 <1% – **CDKN2A** 9q (12) <1% – **BRAF** 7q 34 <1% – **CSF1R** – – – Poor

**MPL** – – – Poor

MDS: myelodysplastic syndrome; CMML: chronic myelomonocytic leukemia; JMML: juvenile myelomonocytic leukemia; AML: acute myeloid leukemia; RCMD: refractory cytopenia with multilineage dysplasia; RA: refractory anemia; RAEB: refractory anemia with excess of blasts; RARS: refractory anemia with ring sideroblasts; RARS-T:

TP53 encodes a cytoplasmic protein p53 that regulates cell growth and death. TP53 mutations have been found mainly in intermediate to high-risk MDS patients [41]. Patients having TP53

20q 13.3 Approximately 1%

12q 24 Approximately 1%

**ATRX** – Rare Associated with

**Frequency Prognostic**

**significance**

Unknown

Unknown

prognosis – Advanced MDS

AML

prognosis – Advanced MDS

AML

– Progression to

– Progression to

**– Associations: phenotypes and MDS types – Application to treatment**

– Normal karyotype predominantly

acquired α-thalassemia, often with severe

anemia

5% of RARS – T

Mouse models of the 5q-syndrome have indicated that a p53-dependent mechanism underlies the pathophysiology of this disorder. Importantly, activation of p53 has been demonstrated in the human 5q-syndrome [43]. Recurrent TP53 mutations have been associated with an increased risk of AML disease evolution and with decreased response to lenalidomide therapy in del (5q) MDS patients [43].

TP53 mutations are usually present years before disease progression. They are associated with p53 overexpression but are not associated with specific clinical manifestations [42]. The presence of TP53 mutations in low-risk MDS with del (5q) contributes to the heterogeneous disease and may significantly affect clinical decision making [42].


**Table 6.** Point mutations in MDS.

In patients with 5q-syndrome, TP53 mutations are present in a small fraction of patients and they cause p53 overexpression subsequently. These aberrant subclones remain quiescent during treatment with lenalidomide and they expand at transformation into acute leukemia [44]. Studies have confirmed that in patient with low-risk MDS having 5q-syndrome, TP53 mutations are associated with strong p53 expression and that p53 positivity is the strongest independent predictor of transformation into AML [45]. Patients with MDS having del (5q) may have mutations other than TP53 such as FOXP1, TP63, JAK2, and MPL mutations [19, 46]. FOXP1 and TP63 mutations may be involved not only in the pathogenesis of the disease, but also they may play a role in the progression into AML [46]. JAK2 and MPL mutations may be found in a small proportion of patients, but their presence does not seem to affect phenotype or progression [19].

Potential new therapeutic agents for del (5q) MDS include the translation enhancer L-leucine, as it may have some efficacy in ribosomopathies. L-leucine has shown increased hemoglobi‐ nization and red cell numbers and reduced developmental defects both in humans and in mouse models [43].

#### **6. Prognostic systems in MDSs**

**Pathway Examples of**

108 Myelodysplastic Syndromes

DNA methylation – DNMT3A

Chromatin modification – ASXL1

Cohesion complex/family

Transcription factors and

MDS: myelodysplastic syndrome.

**Table 6.** Point mutations in MDS.

or progression [19].

pathway

corepressors

Signal transduction – NRAS/KRAS

DNA splicing machinery – SF3B1, – UZAF1

**genetic mutations**

– SRSF1, – PRPF8 – SRSR2/SRSF2, – UTx

– TET2 – IDH1/IDH2

– EZH2

– CBL – JAK2 – NF1 – FLT3

– STAG2 – RAD21 – SMC1A – SMC3

– TP53 – RUNX1 – BCOR1/BCORL1

– CEBPA – ETV6

**Frequency in MDS**

60–70% None

**Application to treatment**

40–50% – DNA methyl transferase inhibitors

– JAK inhibitors – FLT3 inhibitors

20–30% – Deacetylyase inhibitors

20–30% – Kinase inhibitors

10% None

20–40% None

In patients with 5q-syndrome, TP53 mutations are present in a small fraction of patients and they cause p53 overexpression subsequently. These aberrant subclones remain quiescent during treatment with lenalidomide and they expand at transformation into acute leukemia [44]. Studies have confirmed that in patient with low-risk MDS having 5q-syndrome, TP53 mutations are associated with strong p53 expression and that p53 positivity is the strongest independent predictor of transformation into AML [45]. Patients with MDS having del (5q) may have mutations other than TP53 such as FOXP1, TP63, JAK2, and MPL mutations [19, 46]. FOXP1 and TP63 mutations may be involved not only in the pathogenesis of the disease, but also they may play a role in the progression into AML [46]. JAK2 and MPL mutations may be found in a small proportion of patients, but their presence does not seem to affect phenotype

Potential new therapeutic agents for del (5q) MDS include the translation enhancer L-leucine, as it may have some efficacy in ribosomopathies. L-leucine has shown increased hemoglobi‐

– IDH1/IDH2 inhibitors

**(%)**

In MDS, there are several prognostic scoring systems and these include the following: (1) the international prognostic scoring index (IPSS), (2) the revised IPSS (R-IPSS) (**Table 7**), (3) the WHO prognostic scoring system (WPSS), (4) MD Anderson Cancer Center (MDACC) MDS model that includes the global and the lower-risk scoring systems, and (5) the French prog‐ nostic scoring system (FPSS) [30, 31, 33, 47, 48]. The components of the prognostic stratification systems of MDS are as follows: BM blast cells, age, comorbid medical conditions, serum lactic dehydrogenase (LDH), cytogenetics, number of cytopenia, severity of anemia, and high white blood cell (WBC) count [4].


MDS: myelodysplastic syndrome; Hb: hemoglobin; ANC: absolute neutrophil count – indicates not applicable; PLT: platelet.

**Table 7.** (R-IPSS) Revised international prognostic scoring system for MDS.

The IPSS is composed of: blast percentage, karyotype or cytogenetics and the number of cytopenia. The IPSS is classified into low, intemediate-1, intemediate-2, and high-risk score [6, 49]. The R-IPSS model incorporates: BM blasts, cytogenetics, hemoglobin (Hb) level, PLTs, and ANC. The R-IPSS is divided into five risk categories: very low, low, intermediate, high, and very high risks (**Table 7**) [30, 31, 33, 47]. The R-IPSS is an excellent predictor of MDS in the era of disease modifying therapies. The early recognition of patients at high risk of progression to aggressive disease may optimize the timing of treatment before worsening of comorbidities [50]. The precise definition of a prognostic score, such as the R-IPSS, and the probability of leukemia evolution are particularly important in patients with lower-risk MDS in which new approaches including allogeneic HSCT may be addressed in younger patients in a refined manner [50].

The WPSS incorporates the following variables: WHO classification of MDS, cytogenetics, and the need for RBC transfusions [6, 47]. The MD Anderson prognostic model depends on the following factors: age, performance status, prior blood transfusion, WBC and PLT counts, Hb level, BM blasts, and karyotype [47, 49]. The scoring system is divided into four risk categories: low, intermediate-1, intermediate-2, and high [47, 49]. The FPSS includes the following items: the Eastern Cooperative Oncology Group (ECOG) performance status, IPSS cytogenetic risk, the presence of circulating blasts, and packed red blood cell (RBC) transfusion dependency [48]. The prognostic models of MDS are important, as they are used as tools in determining the severity of the illness, the prognosis of MDS, and the best line of management to be considered, that is, supportive care, hypomethylating agents, immunomodulatory drugs or HSCT [49]. The proliferation index (PI) of specific compartments of BM cells is a dynamic parameter that reflects the ongoing rate of production of hematopoietic cells in MDS. It is directly related to the maturation-associated alteration of distinct subgroups of hematopoietic cells in individual patients [4]. Assessment of the PI of nucleated RBCS and other components of BM precursors, such as myeloid CD34+ hematopoietic progenitor cells, could significantly contribute to a better management of MDS. The PI of nucleated RBCS is emerging as an independent prognostic factor for both OS and progression-free survival (PFS) in MDS [4].

#### **7. Anemia and iron overload in MDSs and 5q-syndrome**

Anemia is a very common finding in MDS patients [51, 52]. Packed RBC transfusions are the only therapeutic option in 40% of MDS patients [51, 52]. RBC transfusions are considered in MDS patients when Hb level falls below 8 g/dL and may provide temporary relief of anemic symptoms [51, 52]. Anemia contributes to cardiac dysfunction predominantly in elderly individuals [51]. In MDS patients, anemia can be corrected by the following: (1) RBC transfu‐ sions, (2) administration of hematopoietic growth factors such as erythropoietin, (3) adminis‐ tration of certain drugs such as lenalidomide, cyclosporine-A, and antithymocyte globulin (ATG), and (4) allogeneic HSCT that is the only curative therapeutic approach [51, 52].

Anemia and blood transfusions have significant impact on the quality of life (QOL) of MDS patients [51]. Transfusion dependency is associated with shortened overall and leukemia-free survival in MDS patients [51]. In these patients, transfusion dependency and iron overload have been retrospectively associated with: (1) inferior survival, (2) worse clinical outcome including cardiac, hepatic, and endocrine dysfunction and in some studies, (3) leukemic transformation, and infectious complications [52].

The most serious side effects of regular blood transfusion are elevation of iron blood levels and iron overload, that is, deposition of iron in body tissues [51–54]. Magnetic resonance imaging (MRI) of the heart and liver is an excellent noninvasive diagnostic tool for (1) the assessment of iron overload and (2) monitoring the response to iron chelation therapy [51, 55, 56]. MRI of the heart and liver is superior to the surrogate markers of iron overload such as serum ferritin, liver iron, ventricular ejection fraction, and tissue-related parameter [51]. The diagnostic parameters used for the evaluation of iron overload in MDS are shown in **Table 8** [55]. Serum erythropoietin is a predictive factor for response to therapy with subcutaneous erythropoietin [57]. MDS patients with higher values of erythropoietin have poorer response to the administration of erythropoietin therapy even at higher doses [57].


\* MDS: myelodysplastic syndrome. \* MRI: magnetic resonance imaging.

**Table 8.** Diagnostic parameters used for evaluation of iron overload in MDS.

#### **7.1. Iron overload in low-risk MDS**

The WPSS incorporates the following variables: WHO classification of MDS, cytogenetics, and the need for RBC transfusions [6, 47]. The MD Anderson prognostic model depends on the following factors: age, performance status, prior blood transfusion, WBC and PLT counts, Hb level, BM blasts, and karyotype [47, 49]. The scoring system is divided into four risk categories: low, intermediate-1, intermediate-2, and high [47, 49]. The FPSS includes the following items: the Eastern Cooperative Oncology Group (ECOG) performance status, IPSS cytogenetic risk, the presence of circulating blasts, and packed red blood cell (RBC) transfusion dependency [48]. The prognostic models of MDS are important, as they are used as tools in determining the severity of the illness, the prognosis of MDS, and the best line of management to be considered, that is, supportive care, hypomethylating agents, immunomodulatory drugs or HSCT [49]. The proliferation index (PI) of specific compartments of BM cells is a dynamic parameter that reflects the ongoing rate of production of hematopoietic cells in MDS. It is directly related to the maturation-associated alteration of distinct subgroups of hematopoietic cells in individual patients [4]. Assessment of the PI of nucleated RBCS and other components of BM precursors, such as myeloid CD34+ hematopoietic progenitor cells, could significantly contribute to a better management of MDS. The PI of nucleated RBCS is emerging as an independent prognostic factor for both OS and progression-free survival (PFS) in MDS [4].

110 Myelodysplastic Syndromes

**7. Anemia and iron overload in MDSs and 5q-syndrome**

transformation, and infectious complications [52].

Anemia is a very common finding in MDS patients [51, 52]. Packed RBC transfusions are the only therapeutic option in 40% of MDS patients [51, 52]. RBC transfusions are considered in MDS patients when Hb level falls below 8 g/dL and may provide temporary relief of anemic symptoms [51, 52]. Anemia contributes to cardiac dysfunction predominantly in elderly individuals [51]. In MDS patients, anemia can be corrected by the following: (1) RBC transfu‐ sions, (2) administration of hematopoietic growth factors such as erythropoietin, (3) adminis‐ tration of certain drugs such as lenalidomide, cyclosporine-A, and antithymocyte globulin (ATG), and (4) allogeneic HSCT that is the only curative therapeutic approach [51, 52].

Anemia and blood transfusions have significant impact on the quality of life (QOL) of MDS patients [51]. Transfusion dependency is associated with shortened overall and leukemia-free survival in MDS patients [51]. In these patients, transfusion dependency and iron overload have been retrospectively associated with: (1) inferior survival, (2) worse clinical outcome including cardiac, hepatic, and endocrine dysfunction and in some studies, (3) leukemic

The most serious side effects of regular blood transfusion are elevation of iron blood levels and iron overload, that is, deposition of iron in body tissues [51–54]. Magnetic resonance imaging (MRI) of the heart and liver is an excellent noninvasive diagnostic tool for (1) the assessment of iron overload and (2) monitoring the response to iron chelation therapy [51, 55, 56]. MRI of the heart and liver is superior to the surrogate markers of iron overload such as serum ferritin, liver iron, ventricular ejection fraction, and tissue-related parameter [51]. The diagnostic parameters used for the evaluation of iron overload in MDS are shown in **Table 8** In patients with low-risk MDS, packed RBC transfusion are required to correct anemia. Ultimately, these patients become transfusion dependent [55, 58, 59]. Also, more aggressive disease is usually associated with a high transfusion rate and thus significant transfusion dependency becomes a surrogate marker of aggressive disease [59]. On the long-term, transfusion dependence leads to the development of iron overload, which becomes an important clinical problem, that is associated with an increase in morbidity and mortality [59]. However, in some transfusion-independent low-risk MDS patients, an increased erythro‐ poietic activity results in the suppression of hepcidin and contributes to iron loading [55].

In patients with low-risk MDS who are chronically transfused, transfusion-related morbidity is an emerging challenge [58]. Blood transfusion therapy may lead to organ toxicity due to the formation of nontransferrin bound iron and resulting in oxidative stress. Therefore, in lowrisk MDS patients with longer life expectancy, preventing organ damage due to iron overload is an important concern [59].

Recently, high serum ferritin level has been identified as a prognostic factor for short time to progression to acute leukemia [59]. Transfusion-dependent patients with an isolated erythroid dysplasia and a low risk of leukemic transformation are more likely to develop parenchymal iron overload and its toxicity and hence benefit from iron chelation therapy [56]. In low-risk MDS patients with relatively lower RBC transfusion requirements, T2-MRI is indicated every 10–20 units of packed RBCS in order to evaluate the need to: (1) initiate iron chelation therapy, (2) assess the effectiveness of treatment, and (3) determine the need for dose adjustment [55]. Currently, the initiation of iron chelation therapy is based on: (1) the total number of RBC transfusions and (2) increased serum ferritin in transfusion-dependent patients [55]. Iron chelation therapy is generally recommended for selected patients with low-risk MDS [52–54]. It is reasonable to offer iron chelation therapy to low-risk MDS patients who are at high risk of developing iron overload [55, 59]. Data from multiple retrospective studies have demon‐ strated that iron chelation therapy results in marked survival benefit in patients with low-risk MDS [52, 55].

#### **8. Management of MDSs and 5q-syndrome**

Most patients with MDS are treated with supportive measures due to their old age and comorbid medical conditions [3]. However, there are various therapeutic options for patients with low or intermediate-1 risk MDS and these include the following: (1) blood product transfusions: packed RBCS and PLTs, (2) iron chelation therapy with: deferasirox, deferoxa‐ mine, and deferiprone, (3) erythropoietin with or without granulocyte-CSF, (4) ATG and cyclosporine-A, (5) danazol, (6) pyridoxine, (7) valproic acid, (8) lenalidomide, (9) 5-azaciti‐ dine, (10) decitabine, and (11) low-dose cytarabine [60].

#### **8.1. Iron chelation therapy**

The role of iron chelation therapy in MDS patients with transfusion dependency and iron overload remains a very controversial issue in the management of MDS, mainly due to lack of solid prospective clinical trials [52, 59, 61]. However, case–control studies, retrospective analyses, and phase-II clinical trials have indicated that iron chelation therapy reduces iron overload as measured by serum ferritin and may even prolong overall survival [54].

Iron chelation therapy is indicated in the following categories of patients: (1) patients with frank iron overload; stable or increasing serum ferritin > 1000 ng/mL without signs of active inflammation or liver disease; who are transfusion-dependent at any frequency and have a life expectancy of >1 year, (2) transfusion-dependent patient who receive > 2 units of packed RBCs per month, at any serum ferritin level, and have a life expectancy of > 2 years, except for patients with frank iron deficiency such as chronic gastrointestinal (GIT) bleeding, and (3) in selected patients, iron chelation therapy is considered when life expectancy < 2 years. Examples include: planned curative treatment such as HSCT, massive iron overload with consecutive organ dysfunction or massive iron overload that is judged to significantly reduce QOL [51, 58]. Additional parameters that may influence decision to treat with iron-chelating agents in selected MDS patients include the following: (1) old age, (2) social and mental circumstances, (3) comorbidity and organ dysfunction, and (4) genetic status such as HFE gene mutations [51, 58]. The guidelines of the National Comprehensive Cancer Network **(**NCCN**)** and those of the MDS Foundation for the treatment of iron overload in MDS are illustrated in **Table 9** [52, 55]. The proposed response criteria for iron chelation therapy in MDS are shown in **Table 10** [58].


MDS: myelodysplastic syndrome; HSCT: hematopoietic stem cell transplantation; IPSS: international prognostic scoring index; WHO: world health organization; RA: refractory anemia; RARS: refractory anemia with ring sideroblasts.

**Table 9.** Guidelines for the treatment of iron overload in MDS.

Currently, the initiation of iron chelation therapy is based on: (1) the total number of RBC transfusions and (2) increased serum ferritin in transfusion-dependent patients [55]. Iron chelation therapy is generally recommended for selected patients with low-risk MDS [52–54]. It is reasonable to offer iron chelation therapy to low-risk MDS patients who are at high risk of developing iron overload [55, 59]. Data from multiple retrospective studies have demon‐ strated that iron chelation therapy results in marked survival benefit in patients with low-risk

Most patients with MDS are treated with supportive measures due to their old age and comorbid medical conditions [3]. However, there are various therapeutic options for patients with low or intermediate-1 risk MDS and these include the following: (1) blood product transfusions: packed RBCS and PLTs, (2) iron chelation therapy with: deferasirox, deferoxa‐ mine, and deferiprone, (3) erythropoietin with or without granulocyte-CSF, (4) ATG and cyclosporine-A, (5) danazol, (6) pyridoxine, (7) valproic acid, (8) lenalidomide, (9) 5-azaciti‐

The role of iron chelation therapy in MDS patients with transfusion dependency and iron overload remains a very controversial issue in the management of MDS, mainly due to lack of solid prospective clinical trials [52, 59, 61]. However, case–control studies, retrospective analyses, and phase-II clinical trials have indicated that iron chelation therapy reduces iron

Iron chelation therapy is indicated in the following categories of patients: (1) patients with frank iron overload; stable or increasing serum ferritin > 1000 ng/mL without signs of active inflammation or liver disease; who are transfusion-dependent at any frequency and have a life expectancy of >1 year, (2) transfusion-dependent patient who receive > 2 units of packed RBCs per month, at any serum ferritin level, and have a life expectancy of > 2 years, except for patients with frank iron deficiency such as chronic gastrointestinal (GIT) bleeding, and (3) in selected patients, iron chelation therapy is considered when life expectancy < 2 years. Examples include: planned curative treatment such as HSCT, massive iron overload with consecutive organ dysfunction or massive iron overload that is judged to significantly reduce QOL [51, 58]. Additional parameters that may influence decision to treat with iron-chelating agents in selected MDS patients include the following: (1) old age, (2) social and mental circumstances, (3) comorbidity and organ dysfunction, and (4) genetic status such as HFE gene mutations [51, 58]. The guidelines of the National Comprehensive Cancer Network **(**NCCN**)** and those of the MDS Foundation for the treatment of iron overload in MDS are illustrated in **Table 9** [52, 55]. The proposed response criteria for iron chelation therapy in MDS are shown in **Table 10** [58].

overload as measured by serum ferritin and may even prolong overall survival [54].

MDS [52, 55].

112 Myelodysplastic Syndromes

**8. Management of MDSs and 5q-syndrome**

dine, (10) decitabine, and (11) low-dose cytarabine [60].

**8.1. Iron chelation therapy**


**Table 10.** Proposed response criteria for iron chelation therapy in MDS.

There are three forms of iron-chelating agents, namely (1) oral deferasirox (exjade), (2) oral deferiprone (feriprox), and (3) parenteral deferoxamine (desferal) [54, 58]. The availability of two effective oral chelating agents, deferasirox and deferiprone, has renewed interest in the evaluation of iron chelation therapy in MDS [52].

The beneficial effects of iron chelation therapy in MDS patients having iron overload include significant reduction of: labile plasma iron, nontransferrin-bound iron and reactive oxygen species (ROS) that mediate tissue damage observed in iron overload [52]. Adverse effects of iron chelation therapy include cost and toxicity. Therefore, MDS patients should be initiated on iron chelation therapy after weighing potential risks and benefits for each patient until more definitive data are available [53]. Application of recent advances in the treatment of MDS can reduce or eliminate the need for blood product transfusions thus minimizing the risk of iron overload [54]. Careful attention to iron parameters with early initiation of iron chelation therapy in patients with evidence of transfusion-related iron overload is an important component of high-quality MDS care [61].

#### **8.2. Lenalidomide**

There are 40 genes in the CDR on the long arm of chromosome 5 in MDS with del (5q). Examples of the CDR genes and the effects of lenalidomide on the haplodeficient genes are shown in **Table 11** [13, 62, 63].


MDS: myelodysplastic syndrome.

**Table 11.** Genes in the commonly deleted region and effects of lenalidomide on the haplodeficient genes.

Lenalidomide is a novel thalidomide analog that has enhanced immunomodulatory and antiangiogenic effects and diminished thalidomide-related adverse events [60]. The approved indications of lenalidomide treatment in MDS include the following: (1) patients with del (5q) who have symptomatic transfusion-dependent anemia, (2) lower risk, according to IPSS, MDS patients having 5q- syndrome, and (3) other low-risk and intermediate 1 risk types of MDSs [60, 62].

The important clinical trials of lenalidomide in patients with MDS are shown in **Table 12** [60, 62–66]. Lenalidomide has several mechanisms of action that include the following: (1) promotion of erythropoiesis by inhibition of CD45 protein tyrosine phosphatase and activation of EPO-R/STAT5-signaling pathway, (2) the stimulation of production of certain ILs such as IL-2, IL-10, and interferon (IFN) -δ, (3) inhibition of pro-inflammatory cytokines and chemo‐ kines such as tumor necrosis factor (TNF)-α, IL-12, IL-1*B*, IL-6, monocyte chemotactic pro‐ tein-1, and macrophage anti-inflammatory protein-1α, (4) anti-angiogenic effects of lenalidomide-mediated through endothelial cell migration inhibition, that is, inhibition of bFGF-, VEGF-, and TNF-α-induced endothelial cell migration, (5) immunomodulatory effects, (6) anti-inflammatory properties, (7) direct antineoplastic activity by the inhibition of malig‐ nant clone and upregulation of SPARC gene, (8) direct cytotoxic effect on abnormal del (5q) clones by targeting haploinsufficient genes and their pathways, (9) T-cell activation or stimulation of T-cell proliferation including natural killer (NK) cells number and function and production of multiple cytokines, and (10) inhibition of haplodeficient phosphatases and release of progenitors from p53 arrest [14, 21, 60, 62, 63, 67]. The adverse effects of lenalidomide include the following: (1) myelosuppression: neutropenia, anemia and thrombocytopenia, (2) venous thromboembolism such as deep venous thrombosis in 3.4% of treated patients, (3) infectious complications such as pneumonia, fever and febrile neutropenia, (4) skin rashes, pruritis, and urticaria, (5) GIT upset including nausea and diarrhea, (6) fatigue, muscle cramps, and bone pains, (7) bleeding diathesis, (8) hypokalemia, (9) autoimmune hemolytic anemia, (10) edema formation, and (11) rarely, hypothyroidism and hypogonadism [15, 17, 60, 62, 64, 68]. The mechanisms of resistance to lenalidomide include the following: (1) over expression of PP2A and (2) restoration of p53 expression leading to accumulation of p53 [62].

**8.2. Lenalidomide**

114 Myelodysplastic Syndromes

**Table 11** [13, 62, 63].

RPS 14 Defective ribosomal processing

EGR1 Decrease in tumor suppressors

> Elevated innate immune signaling

Defective G2 – M phase regulation

DIAPH Defective cytoskeleton tumor suppression

MDS: myelodysplastic syndrome.

miR145 miR-146a

CDC 25C1 PP2A

[60, 62].

**Gene Effect of deletion phenotype Effect of**

– Thrombocytopenia

– Leukocytosis – Anemia

– Thrombocytopenia

– Thrombocytosis – Neutropenia – Megakaryocytic dysplasia

G1 and G2 M arrest and apoptosis

**Table 11.** Genes in the commonly deleted region and effects of lenalidomide on the haplodeficient genes.

Clonal dominance Unknown; to be

Lenalidomide is a novel thalidomide analog that has enhanced immunomodulatory and antiangiogenic effects and diminished thalidomide-related adverse events [60]. The approved indications of lenalidomide treatment in MDS include the following: (1) patients with del (5q) who have symptomatic transfusion-dependent anemia, (2) lower risk, according to IPSS, MDS patients having 5q- syndrome, and (3) other low-risk and intermediate 1 risk types of MDSs

The important clinical trials of lenalidomide in patients with MDS are shown in **Table 12** [60, 62–66]. Lenalidomide has several mechanisms of action that include the following: (1) promotion of erythropoiesis by inhibition of CD45 protein tyrosine phosphatase and activation of EPO-R/STAT5-signaling pathway, (2) the stimulation of production of certain ILs such as

SPARC Increased cell adhesion – Anemia

There are 40 genes in the CDR on the long arm of chromosome 5 in MDS with del (5q). Examples of the CDR genes and the effects of lenalidomide on the haplodeficient genes are shown in

**lenalidomide**

in patients with del (5q)

Increased expression in an MDS-related del (5q) cell line

Increased expression in patients with del (5q)

– Direct inhibition of: CDC 25 C – Indirect inhibition of: PP2 A

determined

ex vivo

– Macrocytic anemia Increased expression

Increased expression in MDS CD34+ cells

**Functional effect of lenalidomide**

– Inhibition of proliferation and adhesion

Erythroid response

Reduced proliferation

Possible anti-inflammatory

G1 and G2 M arrest and apoptosis Restoration of

erythropoiesis

Immunomodulatory Antiproliferative

Unknown

Cereblon, an E3 ligase protein, was first described to be the molecular target of lenalidomide in a seminal paper, published in the year 2010, that linked its role to the teratogenic effects of thalidomide in zebrafish and chicks [69, 70]. In 2011, cereblon was found to play a key role in mediating the antiproliferative and immunomodulatory activities of lenalidomide and pomalidomide in multiple myeloma (MM) and T cells, respectively [69, 70]. Thalidomide has been shown to bind and inhibit cereblon and cereblon loss had been found to cause birth defects [71]. Studies on MM cell lines have shown lack of correlation between cereblon expression and sensitivity to lenalidomide. However, in MM cell lines, made resistant to lenalidomide and pomalidomide, cereblon protein was greatly reduced [69–71].

The central role of cereblon as a target of lenalidomide and pomalidomide suggests its potential utility as a predictive biomarker of response or resistance to immunomodulatory drug therapy [69]. The currently available commercial assays that are used in measuring cereblon levels have their own limitations. Therefore, standardization and validation of the techniques used are needed to accurately assess the role of cerebron as a predictive biomarker of the response to immunomodulatory drugs [69].

The serine-thionine kinase, casein kinase 1α (CK1α), is encoded by casein kinase 1 A1 (CSNK1A1) gene [72, 73]. CK1α has been implicated in the biology of del (5q) MDS and has been shown to be a therapeutic target in myeloid malignancies and is therefore an attractive candidate for mediating the effects of lenalidomide in del (5q) MDS [73]. CSNK1A1 gene is a putative tumor suppressor gene located in the CDR at 5q 32 for del (5q) MDS and is expressed at haploinsufficiency levels in MDS with del(5q) [72–74]. Haploinsufficiency of CSNK1A1 leads to hematopoietic stem-cell expansion in mice and may play a role in the initial clonal expansion in patients with 5q-syndrome [43]. CSNK1A1 gene plays a central role in the biology or pathogenesis of del (5q) MDS and is a promising therapeutic target [74]. Lenalidomide induces the ubiquitination of CSNK1A1 by the E3 ubiquitin ligase [CRL4 CRBN] resulting in CSNK1A1 degradation. Lenalidomide significantly alters the protein abundance of three out of five differentiated ubiquitinated proteins [73].

The development of CK1α inhibitors may provide a new therapeutic opportunity in MDS patients with del (5q) and CSNK1A1 mutations [72]. In MDS patients with del (5q), CSNK1A1 mutations have been found in 7.2% of patients and are associated with older age [72]. CSNK1A1 mutations may coexist with ASKL1 but not with p53 mutations. They are usually responsive to lenalidomide and have no independent prognostic impact on overall survival [72].

In del (5q) MDS, lenalidomide-induced degradation of CSNK1A1 below the haploinsufficiency levels induces p53 activity, that is, CSNK1A1 is a negative regulator of p53 [73]. The deletion of genes on chromosome 5q, such as RPS-14, may further sensitize del (5q) cells to p53 activation. This mechanism of activity is consistent with the acquisition of TP53 mutations in del (5q) MDS patients who develop resistance to lenalidomide [73].

Lenalidomide is a potent therapy for low-risk MDS with del (5q) that causes transfusion independency in 67% of patients and complete cytogenetic remission in 45% of patients [44]. However, 50% of patients responding to lenalidomide relapse within 2 years, and 15% of patients achieving cytogenetic response, and 67% of patients not achieving cytogenetic response are at risk of leukemic transformation within 10 years [44].

#### **8.3. The role of HSCT in MDSs**

Allogeneic HSCT is the only known curative therapeutic option for MDS [2, 3, 75–84]. Not only the rates of allogeneic HSCT to treat MDS are continuously increasing, but also survival rates are steadily improving [76, 85]. In patients with MDS, the indications of HSCT are as follows: (1) higher-risk MDS, (2) intermediate-2 MDS, (3) MDS in blast cell crisis, (4) younger patients with MDS having good performance status, and (5) patients with low-risk MDS with poor prognostic features such as: old age, refractory cytopenias and transfusion-dependence [3, 75, 76, 81, 82]. Both blast percentage and percentage of cytogenetically abnormal cells reflect MDS disease burden and predict the outcome of HSCT [86]. Therefore, accurate assessment of MDS disease burden and MDS disease biology based on cytogenetic and molecular profiles is critical to determine the optimal HSCT timing and improve the outcome of HSCT [86]. Incorporation of novel diagnostic techniques such as flow cytometry, molecular cytogenetics and microarray gene expression profiling in the diagnostic algorithms and risk stratification may further optimize therapeutic decisions including the timing of allogeneic HSCT [85].

In patients with MDS undergoing HSCT, predictors of the outcome of HSCT include the following: (1) age, (2) performance status, (3) transfusion dependence, (4) serum erythropoietin level, (5) HSCT-comorbidity index, (6) MK, (7) MDS risk score such as R-IPSS category, and (8) severity of cytopenias [75, 76, 79, 82]. Novel classification schemes for MDS allow for more accurate prognostication and consequently recommendations for HSCT or non-HSCT therapies [78]. MDS disease classification by IPSS, R-IPSS, and WPSS as well as patient characteristics as assessed by HSCT-comorbidity index provide guidance for optimal patient management [81].


or pathogenesis of del (5q) MDS and is a promising therapeutic target [74]. Lenalidomide induces the ubiquitination of CSNK1A1 by the E3 ubiquitin ligase [CRL4 CRBN] resulting in CSNK1A1 degradation. Lenalidomide significantly alters the protein abundance of three out

The development of CK1α inhibitors may provide a new therapeutic opportunity in MDS patients with del (5q) and CSNK1A1 mutations [72]. In MDS patients with del (5q), CSNK1A1 mutations have been found in 7.2% of patients and are associated with older age [72]. CSNK1A1 mutations may coexist with ASKL1 but not with p53 mutations. They are usually responsive to lenalidomide and have no independent prognostic impact on overall survival

In del (5q) MDS, lenalidomide-induced degradation of CSNK1A1 below the haploinsufficiency levels induces p53 activity, that is, CSNK1A1 is a negative regulator of p53 [73]. The deletion of genes on chromosome 5q, such as RPS-14, may further sensitize del (5q) cells to p53 activation. This mechanism of activity is consistent with the acquisition of TP53 mutations in

Lenalidomide is a potent therapy for low-risk MDS with del (5q) that causes transfusion independency in 67% of patients and complete cytogenetic remission in 45% of patients [44]. However, 50% of patients responding to lenalidomide relapse within 2 years, and 15% of patients achieving cytogenetic response, and 67% of patients not achieving cytogenetic

Allogeneic HSCT is the only known curative therapeutic option for MDS [2, 3, 75–84]. Not only the rates of allogeneic HSCT to treat MDS are continuously increasing, but also survival rates are steadily improving [76, 85]. In patients with MDS, the indications of HSCT are as follows: (1) higher-risk MDS, (2) intermediate-2 MDS, (3) MDS in blast cell crisis, (4) younger patients with MDS having good performance status, and (5) patients with low-risk MDS with poor prognostic features such as: old age, refractory cytopenias and transfusion-dependence [3, 75, 76, 81, 82]. Both blast percentage and percentage of cytogenetically abnormal cells reflect MDS disease burden and predict the outcome of HSCT [86]. Therefore, accurate assessment of MDS disease burden and MDS disease biology based on cytogenetic and molecular profiles is critical to determine the optimal HSCT timing and improve the outcome of HSCT [86]. Incorporation of novel diagnostic techniques such as flow cytometry, molecular cytogenetics and microarray gene expression profiling in the diagnostic algorithms and risk stratification may further

In patients with MDS undergoing HSCT, predictors of the outcome of HSCT include the following: (1) age, (2) performance status, (3) transfusion dependence, (4) serum erythropoietin level, (5) HSCT-comorbidity index, (6) MK, (7) MDS risk score such as R-IPSS category, and (8) severity of cytopenias [75, 76, 79, 82]. Novel classification schemes for MDS allow for more accurate prognostication and consequently recommendations for HSCT or non-HSCT

del (5q) MDS patients who develop resistance to lenalidomide [73].

response are at risk of leukemic transformation within 10 years [44].

optimize therapeutic decisions including the timing of allogeneic HSCT [85].

**8.3. The role of HSCT in MDSs**

of five differentiated ubiquitinated proteins [73].

[72].

116 Myelodysplastic Syndromes

**Table 12.** Clinical trials on lenalidomide in MDS.

The age of MDS patients undergoing HSCT has increased significantly over the last 30 years. While HSCT is being carried out in older patients with MDS, this enthusiasm has been over shadowed by the impact of the procedure and its complications, namely graft versus host disease (GVHD) on the QOL and socioeconomic status [78]. Comorbid medical conditions are the major patient characteristics impacting transplantation success. Validation of comorbidity scoring systems has provided the basis for risk assignment to a given patient [78]. HSCT comorbidity index allows estimation of the probability of non-relapse mortality after HSCT [81].

Pre-HSCT serum ferritin > 100 mg/L has been shown to have an adverse impact on OS following HSCT [75]. Adverse consequences of iron overload on the outcome of HSCT include increased risk of septicemia, invasive fungal infections, and sinusoidal obstruction syndrome [75]. BM blast percentage < 5% at the time of HSCT is the major predictor of improved diseasefree survival (DFS) and disease relapse [80]. Prior treatment to decrease blast percentage < 5% prior to allogeneic HSCT is recommended as it has been shown to improve DFS particularly in patients undergoing nonmyeloablative (NMA)-conditioning therapy [80].

The major factors that have a negative effect on relapse-free survival in MDS patients subjected to HSCT are pretransplant karyotype and pre-HSCT BM blast count [76, 81]. Patients with very poor cytogenetics, including MK, have a 10% or less probability of long-term survival [81]. Studies have also shown that a patient with MDS having MK has lower survival rates, higher relapse rates, and higher overall mortality following allogeneic HSCT [76]. The presence of p53, DNMT3A, and TET2 genetic mutations in the pre-HSCT period decreases the probability of post-transplant survival by a factor of 3–4 [76, 81]. On the other hand, SF3B1 mutations are associated with superior leukemia-free survival and OS in MDS patients subjected to alloge‐ neic HSCT [81].

In MDS patients receiving allogeneic HSCT, the stem cell sources are the following: peripheral blood, BM, and umbilical cord blood (UCB) have yielded similar outcomes [80, 83]. Peripheral blood progenitor cells are currently the preferred source of stem cells due to faster engraftment and higher risk of GVHD giving rise to more potent graft versus tumor (GVT) effect [81, 83]. Cord blood cells are typically associated with slow engraftment and hence higher risk of infections and bleeding complications [81]. UCB-derived hematopoietic grafts provide the advantage of transplanting rather immature cells that allows successful HSCT in some patients even in the presence of HLA mismatches [87]. The following forms of allografts are available for MDS patients who are eligible for transplantation: HLA-matched sibling grafts, matched unrelated donor (MUD) allografts, UCB grafts, and HLA-haploidentical donor allografts [78, 83]. The availability of: HLA matched related and unrelated donors, HLA-haploidentical relatives and UCB helps to identify donors for the vast majority of MDS patients [81]. Tradi‐ tionally, transplantation of HLA-haploidentical cells carries an increased risk of graft rejection and an increased risk of GVHD. However, the recently introduced conditioning regimens have reduced the risk of graft rejection and the administration of cyclophosphamide in the early post-transplant period has minimized the risk of GVHD to similar or even lower rates than observed following HLA-matched donor cell [81].

An increased use of unrelated donors and the establishment of protocols for cord blood HSCT and HLA-haploidentical HSCT have made HSCT available for a rapidly growing number of patients [78]. Haploidentical HSCT performed using T-cell replete allografts and posttransplant cyclophosphamide can achieve outcomes equivalent to those of conventional HSCT using HLA-matched related or unrelated donors [87]. The preferred donor for MDS patients undergoing allogeneic HSCT is an HLA-matched sibling or alternatively a fully matched unrelated donor as both have comparable survival rates. However, MUD form of HSCT is associated with higher treatment-related mortality (TRM) [82].

Development of a broad range of conditioning regimens has allowed clinicians to offer HSCT taking into consideration: stage of the disease and patient characteristics [78]. Conventional myeloablative conditioning (MAC) protocols include the following: total body irradiation (TBI), cyclophosphamide, busulfan, and fludarabine, while reduced intensity conditioning (RIC) regimens incorporate low-dose TBI, fludarabine, cyclophosphamide, ATG, and alem‐ tuzumab in various doses and schedules [75]. MAC therapy is associated with lower relapse rate particularly in patients in complete remission or with < 5% blasts. MAC therapy is also associated with increased toxicity and nonrelapse mortality [78, 80]. Long-term survival results in remission following allogeneic HSCT from HLA-matched related or unrelated donor and high-intensity conditioning treatment are as follows: lower-risk MDS: 75%, intermediate-1 MDS: 60%, intermediate-2 MDS: 45% and high-risk MDS: 30% [81]. NMA or RIC conditioning regimens may be considered for MDS patients who are not candidates for MAC regimens due to comorbidities or old age, but such regimens should ideally be used within the context of a clinical trial [82]. Since approximately 75% of MDS patients are > 60 years of age at diagnosis, MAC-allogeneic HSCT can only be offered to a subset of individuals [82]. In patients unfit for MAC therapy, NMA conditioning yields equivalent: TRM, DFS, and OS [80]. For patients with *de novo* MDS aged 60–70 years, the favored therapy varies according to the IPSS risk for patients with low risk and intermediate-1 IPSS risk, nontransplantation approaches are preferred and for patients with intermediate-2 and high-IPSS risk, RIC-allogeneic HSCT offers overall and quality-adjusted survival benefit [88]. In patients with MDS, emphasis should be shifted from high-dose chemotherapy aimed at maximum tumor-cell kill to RIC allogeneic HSCT relying on the donor cell-mediated GVT effect that is most prominent in patients having chronic GVHD in particular in order to eradicate the disease [81]. RIC regimens for allogeneic HSCT have the capacity to result in long-term remissions in MDS patients who are ineligible for conventional allogeneic HSCT [89]. The role of RIC-allogeneic HSCT in MDS patients is to induce chronic GVHD which in turn reduces relapse rate and improves DFS and OS [82, 89].

Autologous HSCT is applicable only to a minority of younger patients with MDS because of difficulty in harvesting adequate numbers of CD34+ cells even in low-risk MDS patients and lack of graft versus leukemia or GVT effect thus resulting in high risk of MDS relapse [3].

#### **8.4. HSCT in lower risk MDS patients**

The age of MDS patients undergoing HSCT has increased significantly over the last 30 years. While HSCT is being carried out in older patients with MDS, this enthusiasm has been over shadowed by the impact of the procedure and its complications, namely graft versus host disease (GVHD) on the QOL and socioeconomic status [78]. Comorbid medical conditions are the major patient characteristics impacting transplantation success. Validation of comorbidity scoring systems has provided the basis for risk assignment to a given patient [78]. HSCT comorbidity index allows estimation of the probability of non-relapse mortality after HSCT

Pre-HSCT serum ferritin > 100 mg/L has been shown to have an adverse impact on OS following HSCT [75]. Adverse consequences of iron overload on the outcome of HSCT include increased risk of septicemia, invasive fungal infections, and sinusoidal obstruction syndrome [75]. BM blast percentage < 5% at the time of HSCT is the major predictor of improved diseasefree survival (DFS) and disease relapse [80]. Prior treatment to decrease blast percentage < 5% prior to allogeneic HSCT is recommended as it has been shown to improve DFS particularly

The major factors that have a negative effect on relapse-free survival in MDS patients subjected to HSCT are pretransplant karyotype and pre-HSCT BM blast count [76, 81]. Patients with very poor cytogenetics, including MK, have a 10% or less probability of long-term survival [81]. Studies have also shown that a patient with MDS having MK has lower survival rates, higher relapse rates, and higher overall mortality following allogeneic HSCT [76]. The presence of p53, DNMT3A, and TET2 genetic mutations in the pre-HSCT period decreases the probability of post-transplant survival by a factor of 3–4 [76, 81]. On the other hand, SF3B1 mutations are associated with superior leukemia-free survival and OS in MDS patients subjected to alloge‐

In MDS patients receiving allogeneic HSCT, the stem cell sources are the following: peripheral blood, BM, and umbilical cord blood (UCB) have yielded similar outcomes [80, 83]. Peripheral blood progenitor cells are currently the preferred source of stem cells due to faster engraftment and higher risk of GVHD giving rise to more potent graft versus tumor (GVT) effect [81, 83]. Cord blood cells are typically associated with slow engraftment and hence higher risk of infections and bleeding complications [81]. UCB-derived hematopoietic grafts provide the advantage of transplanting rather immature cells that allows successful HSCT in some patients even in the presence of HLA mismatches [87]. The following forms of allografts are available for MDS patients who are eligible for transplantation: HLA-matched sibling grafts, matched unrelated donor (MUD) allografts, UCB grafts, and HLA-haploidentical donor allografts [78, 83]. The availability of: HLA matched related and unrelated donors, HLA-haploidentical relatives and UCB helps to identify donors for the vast majority of MDS patients [81]. Tradi‐ tionally, transplantation of HLA-haploidentical cells carries an increased risk of graft rejection and an increased risk of GVHD. However, the recently introduced conditioning regimens have reduced the risk of graft rejection and the administration of cyclophosphamide in the early post-transplant period has minimized the risk of GVHD to similar or even lower rates than

in patients undergoing nonmyeloablative (NMA)-conditioning therapy [80].

[81].

118 Myelodysplastic Syndromes

neic HSCT [81].

observed following HLA-matched donor cell [81].

In patients with low- or intermediate-1-risk MDS, aged 60–79 years, life expectancy following RIC-allogeneic HSCT is about 38 months compared to 77 months in patients not subjected to HSCT, that is, there is no survival benefit of HSCT in this category of patients [81]. Patients with low or very low-risk MDS should ideally be treated with supportive measures and low intensity therapies, such as lenalidomide, erythropoiesis stimulating agents, hypomethylating agents or immunosuppressive therapies rather than allogeneic HSCT [81, 84].

#### **8.5. Road blocks and other unresolved issues related to HSCT in MDSs**

The major road blocks to a universally successful HSCT are relapse of MDS and NRM, often related to GVHD [75, 78, 83]. Allogeneic HSCT in MDS patients can lead to considerable mortality and morbidity mostly as a consequence of toxicity to organs, infectious complications and GVHD [87]. Acute and chronic GVHD are frequent causes of morbidity after HSCT in MDS patients [78]. Additional research is required to prevent GVHD while maintaining the GVT effect [81]. The graft versus dysplasia resulting from allogeneic HSCT and the infusion of donor leukocytes has led to a great understanding of the immunological mechanisms that govern the outcome of HSCT in MDS patients [3]. Post-transplant relapse is a major hurdle to greater success, particularly in patients with high-risk cytogenetics [78]. Pretransplant cytogenetics and BM blasts are the strongest risk factors for post-HSCT relapse [81]. The time interval from allogeneic HSCT until relapse represents a crucial factor to predict response to salvage therapy and survival in patients with high-risk MDS relapsing after allogeneic HSCT [90]. Strategies to reduce relapse and TRM and improve outcome of HSCT include the following: (1) modification of the intensity of conditioning therapy taking into consideration: age, organ function and comorbid medical conditions, (2) pretransplantation strategies that include (A) improvement of remission: (a) hypomethylating agents and/or histone deacetylase inhibitors (HDACs), (b) induction therapy followed by RIC (FLAMSA), and (c) clofarabine and/or cytosine arabinoside. (B) induction of cytogenetic remission by lenalidomide for 5qsyndrome and hypomethylating agents for monosomy 7, and (3) post-transplantation strat‐ egies that include (a) boosting GVL effect by immune enhancers such as lenalidomide, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), anti-PDL1 (programmed death-ligand 1) and adoptive transfer of tumor-reactive T cells and natural killer cells, (b) maintenance with HDACs and/or donor lymphocyte infusion (DLI), and (c) maintenance with lenalidomide and/ or DLI [83, 91].

The following represent the unresolved issues related to HSCT in MDS: (1) timing of the transplant; standard conditioning for younger patients and RIC for older patient with comor‐ bidities, (2) disease status at transplant, (3) pre-HSCT therapy or pretransplant tumor debulk‐ ing with traditional chemotherapeutic agents or the novel DNA hypomethylating drugs, (4) the intensity of the conditioning therapies, (5) stem cell source and alternative donors, (6) optimal therapy for intermediate-risk MDS, and (7) the combination of HSCT with novel therapies such as hypomethylating agents and immunomodulatory drugs [3, 79, 80, 84, 92]. As MDS patients are usually on the old side, QOL is a top priority for most patients, so discussion regarding transplantation in older patients must include not only the acute effects of transplantation but also the delayed effects [81]. Incorporation or integration of novel non-HSCT therapeutic modalities in the overall management of MDS patients undergoing allogeneic HSCT is needed [78].

#### **9. Prognosis in low-risk MDS**

with low or very low-risk MDS should ideally be treated with supportive measures and low intensity therapies, such as lenalidomide, erythropoiesis stimulating agents, hypomethylating

The major road blocks to a universally successful HSCT are relapse of MDS and NRM, often related to GVHD [75, 78, 83]. Allogeneic HSCT in MDS patients can lead to considerable mortality and morbidity mostly as a consequence of toxicity to organs, infectious complications and GVHD [87]. Acute and chronic GVHD are frequent causes of morbidity after HSCT in MDS patients [78]. Additional research is required to prevent GVHD while maintaining the GVT effect [81]. The graft versus dysplasia resulting from allogeneic HSCT and the infusion of donor leukocytes has led to a great understanding of the immunological mechanisms that govern the outcome of HSCT in MDS patients [3]. Post-transplant relapse is a major hurdle to greater success, particularly in patients with high-risk cytogenetics [78]. Pretransplant cytogenetics and BM blasts are the strongest risk factors for post-HSCT relapse [81]. The time interval from allogeneic HSCT until relapse represents a crucial factor to predict response to salvage therapy and survival in patients with high-risk MDS relapsing after allogeneic HSCT [90]. Strategies to reduce relapse and TRM and improve outcome of HSCT include the following: (1) modification of the intensity of conditioning therapy taking into consideration: age, organ function and comorbid medical conditions, (2) pretransplantation strategies that include (A) improvement of remission: (a) hypomethylating agents and/or histone deacetylase inhibitors (HDACs), (b) induction therapy followed by RIC (FLAMSA), and (c) clofarabine and/or cytosine arabinoside. (B) induction of cytogenetic remission by lenalidomide for 5qsyndrome and hypomethylating agents for monosomy 7, and (3) post-transplantation strat‐ egies that include (a) boosting GVL effect by immune enhancers such as lenalidomide, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), anti-PDL1 (programmed death-ligand 1) and adoptive transfer of tumor-reactive T cells and natural killer cells, (b) maintenance with HDACs and/or donor lymphocyte infusion (DLI), and (c) maintenance with lenalidomide and/

The following represent the unresolved issues related to HSCT in MDS: (1) timing of the transplant; standard conditioning for younger patients and RIC for older patient with comor‐ bidities, (2) disease status at transplant, (3) pre-HSCT therapy or pretransplant tumor debulk‐ ing with traditional chemotherapeutic agents or the novel DNA hypomethylating drugs, (4) the intensity of the conditioning therapies, (5) stem cell source and alternative donors, (6) optimal therapy for intermediate-risk MDS, and (7) the combination of HSCT with novel therapies such as hypomethylating agents and immunomodulatory drugs [3, 79, 80, 84, 92]. As MDS patients are usually on the old side, QOL is a top priority for most patients, so discussion regarding transplantation in older patients must include not only the acute effects of transplantation but also the delayed effects [81]. Incorporation or integration of novel non-HSCT therapeutic modalities in the overall management of MDS patients undergoing

agents or immunosuppressive therapies rather than allogeneic HSCT [81, 84].

**8.5. Road blocks and other unresolved issues related to HSCT in MDSs**

or DLI [83, 91].

120 Myelodysplastic Syndromes

allogeneic HSCT is needed [78].

There are several poor prognostic factors in patients with MDS. The poor prognostic factors in MDS in general are listed in **Table 13** [47]. However, in low-risk disease, the following factors have been found to correlate with poor prognosis: (1) severe anemia, (2) transfusion depend‐ ence, (3) poor performance status, (4) older age, (5) number and severity of medical comor‐ bidities, (6) leukocytosis, and (7) elevated level of serum LDH [4].


**Table 13.** Adverse prognostic factors in MDS.

In patients with del (5q), the following factors have been found to be independent predictors of shortened survival: age, transfusion need at diagnosis and dysgranulopoiesis [18, 19].

### **10. Conclusions and future perspectives**

MDSs including 5q-syndrome are often complicated by BM suppression reflected by cytope‐ nias, infectious complications, iron overload and transformation into AML. Management of these disorders includes the following: (1) supportive care that comprises transfusion of blood products, antimicrobials, growth factors, and iron chelation therapy; (2) targeted therapies, such as lenalidomide, and (3) various forms of HSCT. The role of allogeneic HSCT in MDSs is surging as the recently introduced conditioning therapies have allowed application of this curative therapy to older patients and those with comorbid medical conditions. The recent developments in the science of MDSs will allow more advanced targeted therapies to be integrated into the therapeutic algorithms of these disorders.

The role of cereblon as a molecular target for lenalidomide and pomalidomide and that of CK1α in mediating the effects of lenalidomide will ultimately translate into more refined targeted therapies for patients with del (5q) MDS. Also, early incorporation of more pinpointed targeting of clones harboring TP53 mutations and utilization of the translation enhancer, Lleucine, will further improve not only the management but also the outcome of patients with 5q-syndrome.

#### **Author details**

Khalid Ahmed Al-Anazi\*

Address all correspondence to: kaa\_alanazi@yahoo.com

Department of Adult Hematology and Hematopoietic Stem Cell Transplantation, Oncology Center, King Fahad Specialist Hospital, Dammam, Saudi Arabia

#### **References**


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**10. Conclusions and future perspectives**

integrated into the therapeutic algorithms of these disorders.

Address all correspondence to: kaa\_alanazi@yahoo.com

Center, King Fahad Specialist Hospital, Dammam, Saudi Arabia

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5q-syndrome.

122 Myelodysplastic Syndromes

**Author details**

**References**

Khalid Ahmed Al-Anazi\*

MDSs including 5q-syndrome are often complicated by BM suppression reflected by cytope‐ nias, infectious complications, iron overload and transformation into AML. Management of these disorders includes the following: (1) supportive care that comprises transfusion of blood products, antimicrobials, growth factors, and iron chelation therapy; (2) targeted therapies, such as lenalidomide, and (3) various forms of HSCT. The role of allogeneic HSCT in MDSs is surging as the recently introduced conditioning therapies have allowed application of this curative therapy to older patients and those with comorbid medical conditions. The recent developments in the science of MDSs will allow more advanced targeted therapies to be

The role of cereblon as a molecular target for lenalidomide and pomalidomide and that of CK1α in mediating the effects of lenalidomide will ultimately translate into more refined targeted therapies for patients with del (5q) MDS. Also, early incorporation of more pinpointed targeting of clones harboring TP53 mutations and utilization of the translation enhancer, Lleucine, will further improve not only the management but also the outcome of patients with

Department of Adult Hematology and Hematopoietic Stem Cell Transplantation, Oncology

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#### **Chapter 5**

## **Myelodysplastic Disorders, Monosomy 7**

Khalid Ahmed Al-Anazi

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64549

#### **Abstract**

Myelodysplastic syndromes (MDSs) are heterogeneous hematopoietic disorders associated with various degrees of myelosuppression and transformation into acute leukemia. Chromosome 7 abnormalities occur at any age, have several disease associations, and are generally associated with poor outcome. Treatment of the associated disease conditions may have a positive impact on the outcome of certain types of MDSs. For patients eligible for hematopoietic stem cell transplantation (HSCT), allografts are the standard of care, while supportive measures and the use of hypome‐ thylating agents, such as 5-azacytidine and decitabine, constitute the mainstay of management in individuals who are not fit for allogeneic HSCT. However, the use of hypomethylating agents in conjunction with allogeneic HSCT using nonmyeloabla‐ tive conditioning therapies may be an appealing therapeutic option for older patients with comorbid medical conditions.

**Keywords:** myelodysplastic syndrome, monosomy 7, 5-azacytidine, decitabine, hema‐ topoietic stem cell transplantation

#### **1. Introduction**

MDSs are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, dysplastic changes in the peripheral blood and bone marrow (BM), and a variable risk of progression into acute myeloid leukemia (AML) [1–4]. Primary MDS has a bimodal age incidence. It is usually a disease of old age as more than 50% of patients are ≥70 years of age [5]. Primary MDS is less common in the pediatric population and it includes specific pediatric syndromes such as juvenile chronic myeloid leukemia (JCML) and infantile monos‐ omy 7 syndrome [5].

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The clinical, pathologic, and cytogenetic features of primary MDS in younger patients appear to be different from those in elderly individuals suggesting that this may represent a biologi‐ cally different disease [5]. In patients with MDS, with or without abnormal chromosomal karyotype, the type and the quantity of the abnormal karyotype have clinical values in predicting transformation to acute leukemia [6]. The use of granulocyte-monocyte colony stimulating factor (GM-CSF) in congenital BM failure syndromes may induce or accelerate the onset of leukemic transformation [7].

#### **2. Pathogenetic mechanisms in MDSs**

Several mechanisms are involved in the pathogenesis of MDSs and these include: (1) enhance‐ ment of a self-renewal of a hematopoietic stem cell or acquisition of self-renewal in a progenitor cell, (2) enhancement of proliferative capacity in the disease-sustaining clone and/or in its more differentiated progeny, (3) impairment or blockade of differentiation, (4) genetic or epigenetic instability, (5) antiapoptotic mechanisms in the disease-sustaining cells, (6) evasion of the immune system, and (7) suppression of normal hematopoiesis leading ultimately to BM failure [8].

#### **3. Epigenetics in MDSs**

Epigenetics is the heritable alteration in gene expression without DNA sequence change. The primary epigenetic modifiers are DNA methylation and histone modifications, both of which are potentially reversible [9]. DNA methylation plays a major role in tissue- and stage-specific gene regulation and it increases with age. Aberrant methylation of certain promoter regions can occur in diseases particularly cancers and correlates with gene silencing [9]. Epigenetic changes in the form of modification of the transcriptional capacity of the cell via processes such as DNA methylation and histone deacetylation can also alter gene expression impacting disease biology [10].

Advances in the science of epigenetics have led to better understanding of the specific pathogenetic mechanisms underlying MDSs. DNA methylation provides a major epigenetic code of lineage and development-specific genes that control expression of normal cells [8]. The most relevant molecular mediators of the epigenetic state in MDS are gene expression patterns maintained by methylation of cytosine residues in DNA and covalent modification of histones. TET2 status may be a genetic predictor of response to azacytidine, independently of karyotype and holds promise as one of the tools available to help in better selection of patients for treatment [8].

Cancer is characterized by global DNA hypomethylation and regional promoter hyperme‐ thylation of genes [9, 10]. Promoter methylation of CDKN2B [encoding p15INK4b] has been shown to be restricted to the malignant hematological disorders [9, 10]. Several tumor suppressor genes (TSGs) are inactivated by promoter hypermethylation. Potentially reversible silencing of genes, such as CDKN2B, by promoter methylation has been shown to occur in MDS and it increases with disease progression [9, 10].

The clinical, pathologic, and cytogenetic features of primary MDS in younger patients appear to be different from those in elderly individuals suggesting that this may represent a biologi‐ cally different disease [5]. In patients with MDS, with or without abnormal chromosomal karyotype, the type and the quantity of the abnormal karyotype have clinical values in predicting transformation to acute leukemia [6]. The use of granulocyte-monocyte colony stimulating factor (GM-CSF) in congenital BM failure syndromes may induce or accelerate the

Several mechanisms are involved in the pathogenesis of MDSs and these include: (1) enhance‐ ment of a self-renewal of a hematopoietic stem cell or acquisition of self-renewal in a progenitor cell, (2) enhancement of proliferative capacity in the disease-sustaining clone and/or in its more differentiated progeny, (3) impairment or blockade of differentiation, (4) genetic or epigenetic instability, (5) antiapoptotic mechanisms in the disease-sustaining cells, (6) evasion of the immune system, and (7) suppression of normal hematopoiesis leading ultimately to BM failure

Epigenetics is the heritable alteration in gene expression without DNA sequence change. The primary epigenetic modifiers are DNA methylation and histone modifications, both of which are potentially reversible [9]. DNA methylation plays a major role in tissue- and stage-specific gene regulation and it increases with age. Aberrant methylation of certain promoter regions can occur in diseases particularly cancers and correlates with gene silencing [9]. Epigenetic changes in the form of modification of the transcriptional capacity of the cell via processes such as DNA methylation and histone deacetylation can also alter gene expression impacting

Advances in the science of epigenetics have led to better understanding of the specific pathogenetic mechanisms underlying MDSs. DNA methylation provides a major epigenetic code of lineage and development-specific genes that control expression of normal cells [8]. The most relevant molecular mediators of the epigenetic state in MDS are gene expression patterns maintained by methylation of cytosine residues in DNA and covalent modification of histones. TET2 status may be a genetic predictor of response to azacytidine, independently of karyotype and holds promise as one of the tools available to help in better selection of patients for

Cancer is characterized by global DNA hypomethylation and regional promoter hyperme‐ thylation of genes [9, 10]. Promoter methylation of CDKN2B [encoding p15INK4b] has been shown to be restricted to the malignant hematological disorders [9, 10]. Several tumor suppressor genes (TSGs) are inactivated by promoter hypermethylation. Potentially reversible

onset of leukemic transformation [7].

132 Myelodysplastic Syndromes

**3. Epigenetics in MDSs**

disease biology [10].

treatment [8].

[8].

**2. Pathogenetic mechanisms in MDSs**


**Table 1.** .Etiology, risk factors and epidemiological associations of myelodysplastic syndromes.

#### **4. Etiology and associations of MDSs**

MDSs have several etiologies, risk factors, and epidemiological associations as shown in **Table 1** [11–43]. Also, several hereditary diseases predispose to familial forms of MDS/AML as shown in **Table 2** [11, 16, 18, 23, 25, 27, 44–48].

#### **4.1. Familial MDSs**

Familial MDSs are rare diseases. The most common form of familial MDSs is familial platelet disorder, caused by heterozygous germline RUNX1 mutations, which has the propensity to evolve into myeloid malignancy. Many patients lack history of bleeding or thrombocytopenia [44, 46, 47]. Several cases of T-acute lymphoblastic leukemia (ALL) have been reported in patients with inherited RUNX1 mutations [44]. Novel causative mechanisms such as RUNX1 deficiency result in constitutional microdeletions of 21q22 and myelodysplasia associated with telomerase deficiency [44]. Treatment of familial MDS is allogeneic hematopoietic stem cell transplantation (HSCT) but donors have to be screened for deficiency of RUNX1 and deficiency of telomerase [44].

The following genetic mutations have been described in familial MDS/AML: TERC, TERT, CEBPA, GATA2, and RUNX1 [16, 44, 46–48].

#### **4.2. MonoMac syndrome**

MonoMac syndrome is a familial disorder associated with GATA2 deficiency, inherited as autosomal dominant and causes early onset of MDS/AML [47, 48]. Additional acquisitions include: monosomy 7 and ASXL1 mutations. Genetic mutations are detected in dendritic cells, monocytes, natural killer (NK) cells and B-lymphocytes. Many carriers are asymptomatic. The syndrome is associated with severe infectious complications and familial predisposition to cancer. Aggressive therapeutic strategies are needed as the disease has poor outcome [47, 48]. GATA2 mutations have also been described in familial MDS/AML and Emberger syndrome [46–48].

#### **5. Cytogenetic abnormalities in MDSs**

Chromosomal abnormalities are detectable in 40–60% of patients with *de novo* MDS and approximately 90% of patients with secondary therapy-related MDSs (t-MDSs) [1]. The most frequent cytogenetic abnormalities are del(5q), monosomy 7, del(7q), trisomy 8, complex karyotype, and -Y [1]. Chromosome 5 and 7 abnormalities are considered to be the most frequent recurrent genetic abnormalities in myeloid malignancies (MDS and AML) as they occur in 10–20% of myeloid neoplasms [49].


**Table 2.** Familial MDS/AML.

**4. Etiology and associations of MDSs**

as shown in **Table 2** [11, 16, 18, 23, 25, 27, 44–48].

CEBPA, GATA2, and RUNX1 [16, 44, 46–48].

**5. Cytogenetic abnormalities in MDSs**

occur in 10–20% of myeloid neoplasms [49].

**4.1. Familial MDSs**

134 Myelodysplastic Syndromes

of telomerase [44].

[46–48].

**4.2. MonoMac syndrome**

MDSs have several etiologies, risk factors, and epidemiological associations as shown in **Table 1** [11–43]. Also, several hereditary diseases predispose to familial forms of MDS/AML

Familial MDSs are rare diseases. The most common form of familial MDSs is familial platelet disorder, caused by heterozygous germline RUNX1 mutations, which has the propensity to evolve into myeloid malignancy. Many patients lack history of bleeding or thrombocytopenia [44, 46, 47]. Several cases of T-acute lymphoblastic leukemia (ALL) have been reported in patients with inherited RUNX1 mutations [44]. Novel causative mechanisms such as RUNX1 deficiency result in constitutional microdeletions of 21q22 and myelodysplasia associated with telomerase deficiency [44]. Treatment of familial MDS is allogeneic hematopoietic stem cell transplantation (HSCT) but donors have to be screened for deficiency of RUNX1 and deficiency

The following genetic mutations have been described in familial MDS/AML: TERC, TERT,

MonoMac syndrome is a familial disorder associated with GATA2 deficiency, inherited as autosomal dominant and causes early onset of MDS/AML [47, 48]. Additional acquisitions include: monosomy 7 and ASXL1 mutations. Genetic mutations are detected in dendritic cells, monocytes, natural killer (NK) cells and B-lymphocytes. Many carriers are asymptomatic. The syndrome is associated with severe infectious complications and familial predisposition to cancer. Aggressive therapeutic strategies are needed as the disease has poor outcome [47, 48]. GATA2 mutations have also been described in familial MDS/AML and Emberger syndrome

Chromosomal abnormalities are detectable in 40–60% of patients with *de novo* MDS and approximately 90% of patients with secondary therapy-related MDSs (t-MDSs) [1]. The most frequent cytogenetic abnormalities are del(5q), monosomy 7, del(7q), trisomy 8, complex karyotype, and -Y [1]. Chromosome 5 and 7 abnormalities are considered to be the most frequent recurrent genetic abnormalities in myeloid malignancies (MDS and AML) as they

#### **6. Chromosome 7 abnormalities**

Abnormalities involving chromosome 7 occur in approximately 20% of patients with MDS having clonal cytogenetic abnormalities. Abnormalities of chromosome 7 include: (1) total loss of chromosome 7 [monosomy 7], (2) deletion of a segment of the long arm of chromosome 7 [del(7q)], and (3) translocations involving chromosome 7 [2]. However, these cytogenetic anomalies have different prognostic significance [1, 2, 50, 51]. MDS with monosomy 7 has poor prognosis, while isolated del (7q) has a better outcome compared to isolated monosomy 7 [2]. Del(7q) which has distinct clinical and pathological characteristics should no longer be considered in the same prognostic category as monosomy 7 [2]. Also, the prognostic impact of der(7) t(1,7) (q10 or p10) is less adverse once compared to monosomy 7 or del (7q) [1]. In a series of 246 patients with myeloid disorders: monosomy 7 or -7 was the most frequent chromosomal abnormality as it was reported in 51% of patients with secondary myeloid disorders, del(7q) was found in 7% of cases, and partial monosomy was found in 8% of secondary myeloid diseases, while in *de novo* myeloid disorders, monosomy 7 and del(7q) were reported in only 10% of patients [52].

#### **6.1. Disease associations**

Chromosome 7 abnormalities are associated with: (1) de novo and t-MDS, (2) de novo and therapy related AML (t-AML), (3) JCML, (4) juvenile myelomonocytic leukemia [JMML], (5) familial monosomy 7, (6) primary myelofibrosis, (7) Down's syndrome, (8) Fanconi anemia, and (9) lymphoma [50, 51, 53]. Monosomy 7 is the commonest chromosomal abnormality in all of the above conditions except in primary myelofibrosis, where del(7q) is the commonest chromosome 7 anomaly [53]. In adults, chromosome 7 abnormalities are associated with: (1) advanced age, (2) antecedent MDS, and (3) resistance to current therapies [54]. In patients with MDS and AML, chromosome 7 abnormalities usually carry poor prognosis [54].

#### **6.2. Genes on chromosome 7 and their detection**

Genes that have been reported to have microdeletions involving chromosome 7q21.2-q21.3 include: SAMD9, SAMD9L, and HEPACAM2 [55]. The following acquired somatic deletions have also been reported at chromosome 7q36.1: EZH2, CUL1, and TET2 [56, 57]. Examples of additional genetic mutations that have been reported in monosomy 7 and del(7q) include: ASXL1, RUNX1, CBL, ETV6, FAM40B, FAM115A, SEMA3A, LUC7L2, SSPO, NRCAM, GRM8, HYAL4, RABL5, TRIM24, FISI, and CUX1 [51, 57, 58]. Chromosome 7 abnormalities can be detected by conventional cytogenetics or interphase fluorescence *in situ* hybridization (FISH) [59]. Interphase FISH is a very useful method in detecting -7/7q- in patients with MDS. Also, it is more sensitive in detecting chromosome 7 abnormalities than conventional cytogenetics [59]. Refined chromosomal analysis has emerged as a tool that has considerable impact on decision making and development of treatment protocols in patients with MDS and AML [60].

#### **6.3. The commonly deleted segments (CDSs)**

Several studies on MDS and AML specimens with interstitial deletions on chromosome 7 have implicated three putative CDSs at the following chromosome bands: 7q22, 7q34, and 7q35-36. However, 7q22 is the most frequently deleted band in patients with MDS/AML having del(7q) [3]. The following genes in monosomy 7/del(7q) MDS/AML have been reported to be inacti‐ vated or to harbor recurrent genetic mutations such as EZH2, LUC7L2, and CUX1 [3].

The CDS on the long arm of chromosome 7 between 7q22 and 7q36 has been identified to harbor a number of haploinsufficient myeloid TSGs [49, 50, 54]. Loss of function of at least one TSG contributes to disease progression and leukemogenesis or leukemic transformation [50, 54]. It is feasible to somatically delete a large chromosomal segment that is implicated in tumor suppression in hematopoietic cell population *in vitro* [54]. The CDSs that occur at chromosomal bands7q22, 7q34, and 7q35-q36 contain the following genes: TRIM24, SVOPL, ATP6V0A4, TMEM213, KIAA1549, LUC7L2, KLRG2, CLEK2L, HIPK2, TBXAS1, ZC34AV1L, ZC3HAV1, TTC26, UBN2, C7orf55, TPK1, CNTNAP2, MIR548F3, C7orf33, CUL1, and EZH2 [49].

Loss of TP53 is more frequently associated with del5q rather than del7q, while loss of ETV6 is particularly associated with concurrent del(5q) and del(7q) [49]. CUX1, a gene encoding a homeodomain-containing transcription factor, has been identified within the CDS on chro‐ mosome 7 (7q22.1) [61]. CUX1 is expressed at haploinsufficient levels in leukemias with chromosome 7 abnormalities. Haploinsufficiency of CUX1 gave human hematopoietic cells a significant engraftment advantage on transplantation in immunodeficient mice [61].

Monosomy 7 and del(7q) are highly recurrent chromosomal abnormalities in myeloid malig‐ nancies including: AML, *de novo* MDS, and t-MDS/AML [51, 54, 61]. Also, monosomy 7 and del(7q) are common findings in children and adults who develop MDS as a second malignant neoplasm [27]. In t-MDS/AML with chromosome 7 abnormalities, the peak incidence is between 3 and 7 years after cessation of cytotoxic chemotherapy such as alkylating agents [27]. Under such circumstances, monosomy 7 and del(7q) are not equivalent in prognosis and spectrum of disease phenotype [51].

Monosomy 7 and del(7q) are highly prevalent in acquired cytogenetic abnormalities in *de novo* MDS/AML and t-MDS/AML [3]. The proportion of -7/del (7q) cells is markedly increased in hematopoietic stem cell (HSC) and progenitor cell compartments of MDS patients relative to T and B lymphocytes [3]. Recent studies demonstrating quantitative changes in the fre‐ quencies of phenotypic primitive long-term HSCs, common myeloid progenitors, and granulocyte-monocyte progenitors in MDS patients with -7/del(7q) further support the diverse effects on hematopoiesis [3].

After many attempts, mice with 5A3 deletions in the CDS of chromosome band 7q22 have been successfully generated [54]. The 5A3 deleted mice have shown normal hematologic parameters but have not developed myeloid malignancies spontaneously [54]. Animal studies have also shown that heterozygous 5A3 deletion does not accelerate the evolution of leukemia or modulate the responsiveness to antileukemic drugs, while homozygous 5A3 deletions are embryonically lethal [54].

The following 7q genes have been implicated in contributing to leukemogenesis by haploin‐ sufficiency or epigenetic transcriptional repression: SAMD9L, RASA4, dedicator of cytokinesis 4 (DOCK4), and MLL3 [3]. Animal studies have shown that the long-term HSC compartment is expanded in 5A3+/del mice and that the 5A3 deletion partially rescues defective repopulation in GATA2 mutant mice [3]. Studies have also shown that 7q22 deletions are implicated in playing a strong haploinsufficiency role in leukemogenesis [3]. Mutations in DOCK4 gene which is a putative 7q gene have been identified in prostate and ovarian cancers and studies have demonstrated that DOCK4 gene acts as a tumor suppressor [62]. Depletion of DOCK4 levels in MDS stem and progenitor cells leads to erythroid dysplasia by disrupting the action of cytoskeleton in developing red blood cells (RBCs) ultimately leading to dysplastic mor‐ phology of erythroid cells both *in vivo* and *in vitro* [62].

#### **7. Monosomy 7 MDS**

and (9) lymphoma [50, 51, 53]. Monosomy 7 is the commonest chromosomal abnormality in all of the above conditions except in primary myelofibrosis, where del(7q) is the commonest chromosome 7 anomaly [53]. In adults, chromosome 7 abnormalities are associated with: (1) advanced age, (2) antecedent MDS, and (3) resistance to current therapies [54]. In patients with

Genes that have been reported to have microdeletions involving chromosome 7q21.2-q21.3 include: SAMD9, SAMD9L, and HEPACAM2 [55]. The following acquired somatic deletions have also been reported at chromosome 7q36.1: EZH2, CUL1, and TET2 [56, 57]. Examples of additional genetic mutations that have been reported in monosomy 7 and del(7q) include: ASXL1, RUNX1, CBL, ETV6, FAM40B, FAM115A, SEMA3A, LUC7L2, SSPO, NRCAM, GRM8, HYAL4, RABL5, TRIM24, FISI, and CUX1 [51, 57, 58]. Chromosome 7 abnormalities can be detected by conventional cytogenetics or interphase fluorescence *in situ* hybridization (FISH) [59]. Interphase FISH is a very useful method in detecting -7/7q- in patients with MDS. Also, it is more sensitive in detecting chromosome 7 abnormalities than conventional cytogenetics [59]. Refined chromosomal analysis has emerged as a tool that has considerable impact on decision making and development of treatment protocols in patients with MDS and AML [60].

Several studies on MDS and AML specimens with interstitial deletions on chromosome 7 have implicated three putative CDSs at the following chromosome bands: 7q22, 7q34, and 7q35-36. However, 7q22 is the most frequently deleted band in patients with MDS/AML having del(7q) [3]. The following genes in monosomy 7/del(7q) MDS/AML have been reported to be inacti‐ vated or to harbor recurrent genetic mutations such as EZH2, LUC7L2, and CUX1 [3].

The CDS on the long arm of chromosome 7 between 7q22 and 7q36 has been identified to harbor a number of haploinsufficient myeloid TSGs [49, 50, 54]. Loss of function of at least one TSG contributes to disease progression and leukemogenesis or leukemic transformation [50, 54]. It is feasible to somatically delete a large chromosomal segment that is implicated in tumor suppression in hematopoietic cell population *in vitro* [54]. The CDSs that occur at chromosomal bands7q22, 7q34, and 7q35-q36 contain the following genes: TRIM24, SVOPL, ATP6V0A4, TMEM213, KIAA1549, LUC7L2, KLRG2, CLEK2L, HIPK2, TBXAS1, ZC34AV1L, ZC3HAV1, TTC26, UBN2, C7orf55, TPK1, CNTNAP2, MIR548F3, C7orf33, CUL1, and EZH2 [49].

Loss of TP53 is more frequently associated with del5q rather than del7q, while loss of ETV6 is particularly associated with concurrent del(5q) and del(7q) [49]. CUX1, a gene encoding a homeodomain-containing transcription factor, has been identified within the CDS on chro‐ mosome 7 (7q22.1) [61]. CUX1 is expressed at haploinsufficient levels in leukemias with chromosome 7 abnormalities. Haploinsufficiency of CUX1 gave human hematopoietic cells a

Monosomy 7 and del(7q) are highly recurrent chromosomal abnormalities in myeloid malig‐ nancies including: AML, *de novo* MDS, and t-MDS/AML [51, 54, 61]. Also, monosomy 7 and

significant engraftment advantage on transplantation in immunodeficient mice [61].

MDS and AML, chromosome 7 abnormalities usually carry poor prognosis [54].

**6.2. Genes on chromosome 7 and their detection**

136 Myelodysplastic Syndromes

**6.3. The commonly deleted segments (CDSs)**

Monosomy 7 is characterized by (1) lower median age of affected patients than that of 5qsyndrome, (2) severe refractory cytopenias, (3) rapid disease progression, (4) resistance to therapy, and (5) increased susceptibility to infectious complications [52, 63]. Infections encountered in monosomy 7 may be life-threatening and they include (1) bacterial infections: these are the most common types of infections and may be complicated by sepsis, and (2) invasive aspergillosis [63, 64]. Infectious complications in monosomy 7 are caused by neutro‐ penia, dysfunctional neutrophils, and chemotherapy or targeted therapy given to control the disease [64].

In patients having monosomy 7, isolated monosomy 7 occurs in 36% of the cases, monosomy 7 and one additional chromosomal abnormality are encountered in 14% of patients, and monosomy 7 associated with complex cytogenetics in seen in approximately 50% of the cases [63]. Monosomy 7 can be associated with the following chromosomal abnormalities: trisomy 8, chromosome 5 abnormalities, and t(1,7) [63]. Chromosomal microarray analysis is a clinically useful tool in the diagnosis and follow-up of MDS patients with monosomy 7 [65]. In monos‐ omy 7, there is an association between DNA loss and functional impairment or defect of granulocytes [66]. Monosomy 7 is not rare in acute lymphoblastic leukemia as it has been reported in 3–6% of the cases of ALL and in 16% of Philadelphia chromosome positive ALL as it occurs as a secondary anomaly to t(9,22) [52]. Monosomy 7 carries poor prognosis as studies have shown that (1) relapse rate of monosomy 7 at 1 year to be 81%, and (2) event-free survival at 7 years to be 6% [52]. Monosomy 7 does not usually affect lymphoid subpopulations but it is restricted to committed progenitor cells with the capacity to differentiate into mature myeloid cells [67].

Analysis of expression profiles in CD34+ cells from MDS patients with monosomy 7 has shown a malignant phenotype with highly proliferative potential expressing HOX9A, PRAME, BMI-1, PLAB, and BRCA2 (DNA repair gene) [63]. Gene therapy for chronic granulomatous disease has been reported to cause activation of ectopic viral integration site 1 (EVI1) which in turn induces development of genomic instability that ultimately results in clonal progression toward myelodysplasia and monosomy 7 [68].

Conventional chemotherapy in monosomy 7 carries a high risk of early death and poor response. Even if complete remissions are obtained, they are usually short-lived [63]. Targeted therapies such as 5-azacytidine and lenalidomide are more effective than cytotoxic chemo‐ therapy in patients with monosomy 7 MDS. However, lenalidomide is more effective in patients having monosomy 7 and 5q- syndrome. Complete hematological and even cytogenetic responses have been documented in patients with monosomy 7 MDS treated with lenalido‐ mide [63].

In transplant-eligible patients, allogeneic HSCT is the treatment of choice in patients with monosomy 7 [25, 63, 69]. Following allogeneic HSCT, presence of monosomy 7 is a predictor of unfavorable outcome [52].

Masked monosomy 7 refers to monosomy 7 that is detected by FISH but not by conventional cytogenetics. It has been reported in varying frequencies in patients with MDS [70]. Masked monosomy 7 is less common than has been thought and does not seem to carry the same prognostic weight as monosomy 7 diagnosed by metaphase cytogenetics [70].

#### **7.1. Monosomy 7 in children**

MDS is uncommon in children as it accounts for less than 5% of all hematopoietic neoplasms [33, 71]. Viral infections including Epstein-Barr virus (EBV) may contribute to the pathogenesis of MDS by stimulating a preexisting clone and may induce certain genetic mutations [33]. Chromosome 7 abnormalities, monosomy 7 and del (7q), are common cytogenetic abnormal‐ ities in MDS and they are found in 31% of children with myeloid neoplasms [22, 71]. They are characterized by ineffective erythropoiesis, BM dysplasia, and increased risk of leukemic transformation [22]. Monosomy 7 is the most common chromosomal abnormality in children with MDS [33, 71]. In children, monosomy 7 implies poor prognosis because it is associated with high risk of transformation into acute leukemia including ALL [33, 71].

Treatment of children with MDS/AML associated with monosomy 7 with allogeneic HSCT, using a variety of donor types such as sibling donor, unrelated donor, and umbilical cord blood, as well as different sources such as BM and peripheral blood, is an effective therapeutic modality [69]. In patients with more advanced disease, optimization of conditioning therapies may further improve disease-free survival [69]. Graft versus leukemia effect appears to play a major role in leukemia control for some patients and quality of life (QOL) in patients surviving allogeneic HSCT is usually very good [69].

#### **7.2. Familial monosomy 7 syndrome**

Familial monosomy 7 syndrome is a rare familial disorder [44, 45]. It is inherited as autosomal dominant with incomplete penetrance [45]. Familial monosomy 7 can be partial or complete monosomy and it is associated with the following chromosomal abnormalities: trisomy 8, 5q-, and t(1;7) [45]. It has even sex distribution and often presents before the age of 18 years and the median age at diagnosis in 8 years [45]. Allogeneic HSCT in this category of MDS is problematic due to familial predisposition to cancer, hence the prognosis is usually poor [44, 45]. Familial monosomy 7 has several associations including: (1) inherited BM failure syn‐ dromes, (2) secondary MDS/AML, (3) occupational exposure to chemical toxins, (4) exposure to cytotoxic chemotherapy, particularly alkylating agents, (5) Noonan syndrome, (6) Fanconi anemia, and (7) cerebellar ataxia [44, 45]. The cell origin or phenotype is multipotential progenitor cell [45]. The clinical manifestations of familial monosomy 7 syndrome include complications of cytopenias, dysplasia, and acute leukemic transformation in addition to features of the associated disease conditions [44, 45].

#### **7.3. JMML**

In patients having monosomy 7, isolated monosomy 7 occurs in 36% of the cases, monosomy 7 and one additional chromosomal abnormality are encountered in 14% of patients, and monosomy 7 associated with complex cytogenetics in seen in approximately 50% of the cases [63]. Monosomy 7 can be associated with the following chromosomal abnormalities: trisomy 8, chromosome 5 abnormalities, and t(1,7) [63]. Chromosomal microarray analysis is a clinically useful tool in the diagnosis and follow-up of MDS patients with monosomy 7 [65]. In monos‐ omy 7, there is an association between DNA loss and functional impairment or defect of granulocytes [66]. Monosomy 7 is not rare in acute lymphoblastic leukemia as it has been reported in 3–6% of the cases of ALL and in 16% of Philadelphia chromosome positive ALL as it occurs as a secondary anomaly to t(9,22) [52]. Monosomy 7 carries poor prognosis as studies have shown that (1) relapse rate of monosomy 7 at 1 year to be 81%, and (2) event-free survival at 7 years to be 6% [52]. Monosomy 7 does not usually affect lymphoid subpopulations but it is restricted to committed progenitor cells with the capacity to differentiate into mature

Analysis of expression profiles in CD34+ cells from MDS patients with monosomy 7 has shown a malignant phenotype with highly proliferative potential expressing HOX9A, PRAME, BMI-1, PLAB, and BRCA2 (DNA repair gene) [63]. Gene therapy for chronic granulomatous disease has been reported to cause activation of ectopic viral integration site 1 (EVI1) which in turn induces development of genomic instability that ultimately results in clonal progression

Conventional chemotherapy in monosomy 7 carries a high risk of early death and poor response. Even if complete remissions are obtained, they are usually short-lived [63]. Targeted therapies such as 5-azacytidine and lenalidomide are more effective than cytotoxic chemo‐ therapy in patients with monosomy 7 MDS. However, lenalidomide is more effective in patients having monosomy 7 and 5q- syndrome. Complete hematological and even cytogenetic responses have been documented in patients with monosomy 7 MDS treated with lenalido‐

In transplant-eligible patients, allogeneic HSCT is the treatment of choice in patients with monosomy 7 [25, 63, 69]. Following allogeneic HSCT, presence of monosomy 7 is a predictor

Masked monosomy 7 refers to monosomy 7 that is detected by FISH but not by conventional cytogenetics. It has been reported in varying frequencies in patients with MDS [70]. Masked monosomy 7 is less common than has been thought and does not seem to carry the same

MDS is uncommon in children as it accounts for less than 5% of all hematopoietic neoplasms [33, 71]. Viral infections including Epstein-Barr virus (EBV) may contribute to the pathogenesis of MDS by stimulating a preexisting clone and may induce certain genetic mutations [33]. Chromosome 7 abnormalities, monosomy 7 and del (7q), are common cytogenetic abnormal‐ ities in MDS and they are found in 31% of children with myeloid neoplasms [22, 71]. They are

prognostic weight as monosomy 7 diagnosed by metaphase cytogenetics [70].

myeloid cells [67].

138 Myelodysplastic Syndromes

mide [63].

of unfavorable outcome [52].

**7.1. Monosomy 7 in children**

toward myelodysplasia and monosomy 7 [68].

JMML is a rare clonal MDS/myeloproliferative neoplasm (MPN) of young children [44, 45, 72, 73]. It has also been described as juvenile CML and was formerly grouped in the French-American-British (FAB) classification of MDS [74]. Without treatment, the 10-year overall survival (OS) of patients with JMML is 6% [74]. Allogeneic HSCT is the only curative therapy for children with JMML [72, 74]. Studies have also shown that event-free survival is 52% at 5 years post-HSCT [72]. Also, relapse is expected to occur in 50% of transplanted patients [72, 73]. Treatment options of relapsed JMML after the first HSCT include: (1) withdrawal of immunosuppressive therapy and/or donor lymphocyte infusion, and (2) second allogeneic HSCT, which may be the treatment of choice in such situations [72]. The major causes of HSCT failure in patients with JMML are treatment-related mortality and relapse [74].

#### **8. Anemia in MDS**

Severe anemia should be considered a major criterion for deciding not only the type but also the timing of therapeutic interventions in patients with MDS [75]. Once anemia is symptomatic, transfusion of packed RBCs is the mainstay of therapy in MDS [76]. The redistribution of transfusion iron from reticuloendothelial cells to parenchymal cells is modulated by hepcidin. Ineffective erythropoiesis has a suppressive effect on hepcidin production and hence increases iron redistribution [76].

#### **9. Iron overload in high-risk MDS**

Transfusion history should be considered in transplantation decision making in patients with MDS because pre-HSCT transfusion history and serum ferritin levels have been shown to have significant prognostic value in patients with MDS undergoing allogeneic HSCT [77]. Elevated serum ferritin and elevated liver iron content in patients with MDS and acute leukemia prior to HSCT are associated with inferior post-HSCT survival [78]. Studies have shown that transfusion dependency is independently associated with (1) reduced overall survival, (2) increased nonrelapse mortality (NRM), and (3) increased risk of acute graft versus host disease (GVHD) in patients with MDS undergoing allogeneic HSCT [77].

In patients with high-risk MDS, iron overload has adverse consequences on the outcome of HSCT as it has been associated with (1) increased transfusion-related mortality, (2) infectious complications, and (3) AML progression [79]. Iron chelation therapy in patients with higher risk MDS should be considered to possibly (1) reduce infectious complications, (2) delay leukemic transformation, and (3) improve the outcome of HSCT [80].

Nuclear factor-kappaB (NF-kB) is key regulator of many cellular processes and its impaired activity has been described in different myeloid malignancies including MDS [81]. NF-kB inhibition by deferasirox could prove to be an important therapeutic option in higher risk MDS patients by targeting blast cells in which increased NF-kB activity has been extensively demonstrated thus acting as a possible enhancer of chemosensitivity of the malignant clone [81].

#### **10. Management of MDS**

The following therapeutic modalities are available for patients with MDS: (1) supportive measures: packed RBCs and platelet (PLT) transfusions, antimicrobial therapy, and hemato‐ poietic growth factors, (2) drug therapies including novel agents such as lenalidomide, azacytidine, and decitabine, and (3) various forms of HSCT [82].

#### **10.1. Epigenetic therapies of MDS**

**8. Anemia in MDS**

140 Myelodysplastic Syndromes

iron redistribution [76].

[81].

**10. Management of MDS**

**9. Iron overload in high-risk MDS**

(GVHD) in patients with MDS undergoing allogeneic HSCT [77].

leukemic transformation, and (3) improve the outcome of HSCT [80].

azacytidine, and decitabine, and (3) various forms of HSCT [82].

Severe anemia should be considered a major criterion for deciding not only the type but also the timing of therapeutic interventions in patients with MDS [75]. Once anemia is symptomatic, transfusion of packed RBCs is the mainstay of therapy in MDS [76]. The redistribution of transfusion iron from reticuloendothelial cells to parenchymal cells is modulated by hepcidin. Ineffective erythropoiesis has a suppressive effect on hepcidin production and hence increases

Transfusion history should be considered in transplantation decision making in patients with MDS because pre-HSCT transfusion history and serum ferritin levels have been shown to have significant prognostic value in patients with MDS undergoing allogeneic HSCT [77]. Elevated serum ferritin and elevated liver iron content in patients with MDS and acute leukemia prior to HSCT are associated with inferior post-HSCT survival [78]. Studies have shown that transfusion dependency is independently associated with (1) reduced overall survival, (2) increased nonrelapse mortality (NRM), and (3) increased risk of acute graft versus host disease

In patients with high-risk MDS, iron overload has adverse consequences on the outcome of HSCT as it has been associated with (1) increased transfusion-related mortality, (2) infectious complications, and (3) AML progression [79]. Iron chelation therapy in patients with higher risk MDS should be considered to possibly (1) reduce infectious complications, (2) delay

Nuclear factor-kappaB (NF-kB) is key regulator of many cellular processes and its impaired activity has been described in different myeloid malignancies including MDS [81]. NF-kB inhibition by deferasirox could prove to be an important therapeutic option in higher risk MDS patients by targeting blast cells in which increased NF-kB activity has been extensively demonstrated thus acting as a possible enhancer of chemosensitivity of the malignant clone

The following therapeutic modalities are available for patients with MDS: (1) supportive measures: packed RBCs and platelet (PLT) transfusions, antimicrobial therapy, and hemato‐ poietic growth factors, (2) drug therapies including novel agents such as lenalidomide, Epigenetic therapies cause potentially reversible epigenetic changes that can alter gene expression patterns [83]. Epigenetic therapies in MDS include (1) histone deacetylase inhibi‐ tors, and (2) hypomethylating agents, such as azacytidine and decitabine, that inhibit the DNA methyltransferase enzymes (DNMT) [83].

Epigenetic silencing is a universal mechanism of gene inactivation in malignant cells, probably exceeding mutational events. Recent therapeutic approaches targeting the aberrant epigenome of cancer has been developed [84]. The hypomethylating agents, azacytidine and decitabine, have shown remarkable activity in older individuals with higher risk MDSs including patients with poor-risk cytogenetic profiles. Translational studies performed on BM biopsies obtained from MDS patients with both azanucleoside demethylating agents have indicated that both azanucleosides can revert the aberrant hypermethylation state *in vivo* [84].

#### **10.2. Hypomethylating agents**

The azanucleosides, 5-azacytidine and decitabine, were originally synthesized more than 50 years ago in order to be used as classical cytotoxic agents [85–88]. Azacytidine was first described by Sorm in 1964 as a cancerostatic agent [89]. Both hypomethylating agents, 5 azacytidine and decitabine, had demonstrated activity against lymphoid leukemic cells as well as hemopoietic tissues in experimental leukemia mice models [90–93].

#### *10.2.1. 5-Azacytidine*

Azacytidine is a pyrimidine nucleoside analog that differs from cytosine by the presence of nitrogen, rather than ring carbon, at position 5 [8, 9]. It was first manufactured in Europe in the 1960s [8, 9]. Azacytidine is a DNA methyltransferase inhibitor (DMTI) that has *in vitro* and *in vivo* demethylating effects [9]. The hypomethylating effects of azacytidine appear to primarily depend on the structural alternations at position 5 [8]. Azacytidine was the first hypomethylating agent to be approved by the Food and Drug Administration (FDA) in United States of America for the treatment of all subtypes of MDS in May 2009 [8, 10, 94]. In patients with high-risk MDS, the benefits of azacytidine therapy on survival compared to conventional chemotherapy have not been established outside clinical trials [95, 96]. Despite the wide spread use of azacytidine in the treatment of high-risk MDSs, there is lack of improvement in longterm survival. Therefore, identification of predicting factors of response and survival is mandatory [95, 96].

Hypomethylating agents or azanucleosides are becoming the standard therapy for patients with higher-risk MDSs [97]. Patients with high-risk MDSs treated with azanucleosides have a median overall survival of 11–16 months, so they should be strongly considered for upfront allogeneic HSCT or experimental therapies [97]. In patients with high-risk MDSs planned for allogeneic HSCT, azacytidine treatment may be valuable in stabilizing the disease and preventing relapse [98]. Additionally, pretransplant administration of azacytidine does not adversely affect transplant outcome [98]. Preemptive azacytidine therapy has an acceptable safety profile and can substantially prevent or at least delay relapse in patients with MDS or AML with minimal residual disease after allogeneic HSCT [99].


*Abbreviations*: MDS, myelodysplastic syndrome; IV, intravenous; CALG-B, cancer and leukemia group B; SC, subcutaneous.

**Table 3.** Phase I and phase II CALG-B clinical trials on azacytidine in MDS.

The outcome of patients with high-risk MDSs after failure of azacytidine treatment is generally poor [100]. After failure of azacytidine treatment, the options are rather limited to (1) best supportive care in patients unfit for allogeneic HSCT, and (2) allogeneic HSCT and investiga‐ tional agents in patients who are eligible for such therapies [100]. Mechanisms of action of 5 azacytidine are multifactorial and they include (1) demethylation of several key genes, that is, reduction of DNA methylation by inhibition of methyltransferase enzymes, (2) cytotoxic action by inhibition of protein translation, and (3) enhancement of apoptosis [8–10]. In patients with MDSs, 5-azacytodine is indicated in (1) high-risk MDSs, and (2) intermediate 2 risk MDSs [8, 10, 95, 97, 100–102]. The side effects of azacytidine therapy include myelosuppression (leuco‐ penia, anemia, and thrombocytopenia); gastrointestinal (GIT) upset (nausea, vomiting, diarrhea, and constipation); injection site reactions and erythema; serum sickness-like illness; abnormal liver function tests; fatigue; weakness; lethargy; anorexia; headache; arthralgias; febrile neutropenia; cytomegalovirus infection; and pneumonia [9, 10, 99, 101, 103].

The effects of azacytidine in patients with MDSs include (1) prolongation of survival, (2) improvement in QOL, and (3) delayed leukemic transformation [8, 10, 95, 101, 102]. Responses to azacytidine according to karyotypes are as follows: (1) excellent responses are expected in patients with normal cytogenetics, (2) durable remission and 80% response rate are expected in patients having chromosome 7 abnormalities as the sole karyotypic abnormalities, and (3) good early responses but early relapses in patients with trisomy 8 [9]. Predictors of positive responses to DMTIs is include (1) doubling of PLT count, (2) mutated TET2, (3) mutated EZH2, (4) Phosphoinositide-phospholipase C beta hypomethylation, and (5) low serum level of micro-RNA-21 [96, 104, 105]. Predictors of poor response to DMTIs include (1) BM blasts >15%, (2) previous therapy, (3) transfusion dependency, (4) grade 3 marrow fibrosis, (5) mutated p53, (6) abnormal karyotype of complex cytogenetics, (7) high serum level of micro-RNA-21, and (8) increased cytidine deaminase expression of activity in males [96, 104, 106].

safety profile and can substantially prevent or at least delay relapse in patients with MDS or

**Name of trial CALG-B/8421 trial CALG-B/8921 trial**

continuous IV infusion over 7 days

SC administration 100 mg/m2

Phase Phase I Phase II

every 28 daysdose escalated to 150 mg/m2

*Abbreviations*: MDS, myelodysplastic syndrome; IV, intravenous; CALG-B, cancer and leukemia group B; SC,

febrile neutropenia; cytomegalovirus infection; and pneumonia [9, 10, 99, 101, 103].

The effects of azacytidine in patients with MDSs include (1) prolongation of survival, (2) improvement in QOL, and (3) delayed leukemic transformation [8, 10, 95, 101, 102]. Responses to azacytidine according to karyotypes are as follows: (1) excellent responses are expected in patients with normal cytogenetics, (2) durable remission and 80% response rate are expected in patients having chromosome 7 abnormalities as the sole karyotypic abnormalities, and (3) good early responses but early relapses in patients with trisomy 8 [9]. Predictors of positive responses to DMTIs is include (1) doubling of PLT count, (2) mutated TET2, (3) mutated EZH2, (4) Phosphoinositide-phospholipase C beta hypomethylation, and (5) low serum level of micro-RNA-21 [96, 104, 105]. Predictors of poor response to DMTIs include (1) BM blasts >15%,

The outcome of patients with high-risk MDSs after failure of azacytidine treatment is generally poor [100]. After failure of azacytidine treatment, the options are rather limited to (1) best supportive care in patients unfit for allogeneic HSCT, and (2) allogeneic HSCT and investiga‐ tional agents in patients who are eligible for such therapies [100]. Mechanisms of action of 5 azacytidine are multifactorial and they include (1) demethylation of several key genes, that is, reduction of DNA methylation by inhibition of methyltransferase enzymes, (2) cytotoxic action by inhibition of protein translation, and (3) enhancement of apoptosis [8–10]. In patients with MDSs, 5-azacytodine is indicated in (1) high-risk MDSs, and (2) intermediate 2 risk MDSs [8, 10, 95, 97, 100–102]. The side effects of azacytidine therapy include myelosuppression (leuco‐ penia, anemia, and thrombocytopenia); gastrointestinal (GIT) upset (nausea, vomiting, diarrhea, and constipation); injection site reactions and erythema; serum sickness-like illness; abnormal liver function tests; fatigue; weakness; lethargy; anorexia; headache; arthralgias;

Complete response 5 patients (12%) 8 patients (12%) Partial response 11 patients (25%) 10 patients (15%) Improvement 5 patients (12%) 18 patients (27%) Total response 21 Patients (49%) 36 patients (53%)

Number of patients evaluated 43 68

AML with minimal residual disease after allogeneic HSCT [99].

Initial dose: 75 mg/m2

**Table 3.** Phase I and phase II CALG-B clinical trials on azacytidine in MDS.

Azacytidine therapy IV administration

subcutaneous.

142 Myelodysplastic Syndromes


*Abbreviations*: MDS, myelodysplastic syndrome; AML, acute myeloid leukemia; IPSS, International Prognostic Scoring System; CALG-B, cancer and leukemia group B; AZA, azacytidine HI: hematological improvement.

**Table 4.** Phase III randomized controlled clinical trials on azacytidine.

In patients with high-risk MDSs and AML, the combination of 5-azacytidine, valproic acid, and all-trans retinoic acid (ATRA) are safe and they are active and associated with induc‐ tion of global DNA hypomethylation and histone acetylation [107, 108]. Lessons learned from clinical experience with hypomethylating agents include (1) in the majority of treated patients, the beneficial effects are only noted after approximately four cycles of therapy, (2) the achievement of hematological improvement is sufficient to ensure prolonged OS, (3) in

almost all patients, interruption of treatment induces relapse, (4) patients who relapse after treatment or who are refractory to therapy have extremely limited survival, and (5) pa‐ tients with complex karyotype involving monosomy 7 or monosomy 5 have negligible survival advantage from hypomethylating agents despite achievement of response [96]. Clinical phase I, II, and III trials on the use of azacytidine in patients with MDSs are shown in **Tables 3** and **4** [9, 10, 101, 103, 109]. Investigational agents that can be used in the treatment of MDSs in case of failure of hypomethylating agents include (1) rigosertib, (2) sapacita‐ bine, (3) clofarabine, and (4) BCL2 inhibitors (proapoptotic drug therapy) including ABT-737 and ABT-199 [96, 110].

Conclusion that can be drawn from phase III trials on azacytidine include: (1) in CALG-B 9221 trial: compared to best supportive care (BSC), azacytidine therapy resulted in (a) significantly higher response rates, (b) improved QOL, (c) improved survival, and (d) reduced risk of leukemic transformation; and (2) in AZA-001 trial: compared to conventional therapy, that Included BSC, low dose cytarabine ± intensive chemotherapy, azacytidine increased OS in patients with high-risk MDS [101, 109]. In patients with chromosome 7 abnormalities [mon‐ osomy 7 and del(7q)], the median survival was 13.1 months in patients treated with azacytidine compared to 4.6 months in patients who received conventional therapies [101].

#### *10.2.2. Decitabine*

Decitabine (5-aza-2-deoxycytidine) inhibits DNMT. It was approved by the FDA in the United States for the treatment of MDS in the year 2006 [83, 111, 112]. It is postulated that initially the drug and the DNMT enzymes become attached, then the outcome will be: (1) enzyme degra‐ dation resulting in low DNMT levels, and (2) ultimately achievement of hypomethylation [83]. Although decitabine antitumor activity is not fully understood, there are several possible mechanisms of action that include (1) induction of hypomethylation or reversal of cancerassociated hypermethylation effects, (2) reactivation of genes responsible for cellular differ‐ entiation, (3) stimulation or induction of immune responses, (4) induction of DNA damage pathways or apoptotic response pathways, that is, induction of changes in the rates of apoptosis, and (5) augmentation of stem cell renewal [83, 105]. Various doses, schedules, and even routes of administration have been used: 10, 15, or 20 mg/m2 intravenously (IV) or subcutaneously (SC) for 3–5 days, each cycle for at least four cycles that are given at 4- to 6 week intervals [83, 111, 112].

Although it has been used in the treatment of all FAB subtypes of MDS, the specific indications are as follows: (1) intermediate 1, intermediate 2, and high-risk MDS, (2) *de novo* and secondary MDS, including t-MDS, (3) MDS transforming into AML, in individuals unfit for intensive cytotoxic chemotherapy, as upfront therapy, (4) treatment of MDS refractory to lenalidomide, (5) debulking treatment prior to HSCT in high-risk patients, and (6) patients with chronic myelomonocytic leukemia (CMML) [83, 105, 111]. The adverse effects of decitabine therapy include (1) myelosuppression leading to febrile neutropenia, sepsis, pneumonia, and fungal infections, (2) gastrointestinal effects including nausea, vomiting, diarrhea, and mucositis, (3) hair loss, skin rashes, fatigue, and bleeding, (4) renal failure, (5) cardiovascular complications are uncommon, and (6) pleural effusions and acute lung injury [83, 112, 113]. Encountering myelosuppression that requires decitabine dose modification may truly indicate response to therapy [112].

Despite the efficacy of dectiabine therapy, there are no known definitive predictors of response. However, in patients with high-risk MDS treated with decitabine, high expression of human equilibrative nucleoside transporter-1 (hENT-1) gene appears to predict a good response to decitabine therapy and is associated with prolonged survival [114]. Patients with chromosome 7 abnormalities usually respond more favorably to continuous IV infusion of low-dose decitabine than to conventional chemotherapy with low-dose cytarabine [115]. Results of clinical trials on decitabine are shown in **Table 5** [83, 84, 111, 113]. Unfortunately, there is no head-to-head comparison with 5-azacytidine. Also, decitabine has not shown a statistically significant evidence of prolonged survival benefit in prospective studies. In addition, the role of decitabine after HSCT needs further evaluation [83].

#### *10.2.3. Rigosertib (ON01910.Na)*

almost all patients, interruption of treatment induces relapse, (4) patients who relapse after treatment or who are refractory to therapy have extremely limited survival, and (5) pa‐ tients with complex karyotype involving monosomy 7 or monosomy 5 have negligible survival advantage from hypomethylating agents despite achievement of response [96]. Clinical phase I, II, and III trials on the use of azacytidine in patients with MDSs are shown in **Tables 3** and **4** [9, 10, 101, 103, 109]. Investigational agents that can be used in the treatment of MDSs in case of failure of hypomethylating agents include (1) rigosertib, (2) sapacita‐ bine, (3) clofarabine, and (4) BCL2 inhibitors (proapoptotic drug therapy) including ABT-737

Conclusion that can be drawn from phase III trials on azacytidine include: (1) in CALG-B 9221 trial: compared to best supportive care (BSC), azacytidine therapy resulted in (a) significantly higher response rates, (b) improved QOL, (c) improved survival, and (d) reduced risk of leukemic transformation; and (2) in AZA-001 trial: compared to conventional therapy, that Included BSC, low dose cytarabine ± intensive chemotherapy, azacytidine increased OS in patients with high-risk MDS [101, 109]. In patients with chromosome 7 abnormalities [mon‐ osomy 7 and del(7q)], the median survival was 13.1 months in patients treated with azacytidine

Decitabine (5-aza-2-deoxycytidine) inhibits DNMT. It was approved by the FDA in the United States for the treatment of MDS in the year 2006 [83, 111, 112]. It is postulated that initially the drug and the DNMT enzymes become attached, then the outcome will be: (1) enzyme degra‐ dation resulting in low DNMT levels, and (2) ultimately achievement of hypomethylation [83]. Although decitabine antitumor activity is not fully understood, there are several possible mechanisms of action that include (1) induction of hypomethylation or reversal of cancerassociated hypermethylation effects, (2) reactivation of genes responsible for cellular differ‐ entiation, (3) stimulation or induction of immune responses, (4) induction of DNA damage pathways or apoptotic response pathways, that is, induction of changes in the rates of apoptosis, and (5) augmentation of stem cell renewal [83, 105]. Various doses, schedules, and

subcutaneously (SC) for 3–5 days, each cycle for at least four cycles that are given at 4- to 6-

Although it has been used in the treatment of all FAB subtypes of MDS, the specific indications are as follows: (1) intermediate 1, intermediate 2, and high-risk MDS, (2) *de novo* and secondary MDS, including t-MDS, (3) MDS transforming into AML, in individuals unfit for intensive cytotoxic chemotherapy, as upfront therapy, (4) treatment of MDS refractory to lenalidomide, (5) debulking treatment prior to HSCT in high-risk patients, and (6) patients with chronic myelomonocytic leukemia (CMML) [83, 105, 111]. The adverse effects of decitabine therapy include (1) myelosuppression leading to febrile neutropenia, sepsis, pneumonia, and fungal infections, (2) gastrointestinal effects including nausea, vomiting, diarrhea, and mucositis, (3) hair loss, skin rashes, fatigue, and bleeding, (4) renal failure, (5) cardiovascular complications are uncommon, and (6) pleural effusions and acute lung injury [83, 112, 113]. Encountering

intravenously (IV) or

compared to 4.6 months in patients who received conventional therapies [101].

even routes of administration have been used: 10, 15, or 20 mg/m2

and ABT-199 [96, 110].

144 Myelodysplastic Syndromes

*10.2.2. Decitabine*

week intervals [83, 111, 112].

Rigosertib is a multikinase inhibitor that inhibits both the phosphoinositide 3 kinase and the polo-like kinase pathways [116–120]. It inhibits the cell-cycle progression by selectively inducing a mitotic arrest and apoptosis in cancer cells [116, 118–120]. Recently, it has been highlighted as a novel anticancer agent for the treatment of MDS. Rigosertib has shown activity in the following malignancies: (1) mantle cell lymphoma, (2) chronic lymphocytic leukemia, and (3) MDS [118]. In MDS, rigosertib has several mechanisms of action that include: (a) upregulation of genes related to microtubule kinetics, (b) downregulation of the mRNA degradation system, that is, suppression of nonsense mRNA decay (NMD) gene, (c) suppres‐ sion of cyclin-D1 in BM CD34+ cells in MDS patients with trisomy 8 and monosomy 7, and (d) induction of cell death by inhibition of PI3kinase/Akt pathway and DNA damage-induced G2/ M arrest, that is, induction of mitotic arrest and apoptosis in myeloblasts while sparing normal cells [118–120].

Rigosetib has shown efficacy in all morphologic, prognostic risk and cytogenetic subgroups of MDS and has produced complete responses in some patients [121]. It has shown activity in high-risk MDS patients and in those having monosomy 7 and trisomy 8. It has produced the following beneficial effects: (1) decrease in BM blasts, (2) improvement in hematopoiesis, (3) inhibition of cyclin D1 accumulation, and (4) decrease in trisomy 8 and monosomy 7 aneu‐ ploidy [116, 119, 121]. However, a randomized controlled, phase 3, clinical trial that had been performed in 74 institutions in Europe and the United States on the use of rigosertib in patients with high-risk MDS after failure of hypomethylating agents did not show significant OS compared to best supportive care [117]. The drug is available in oral and injectable formula‐ tions [120, 121]. Although rigosertib has exhibited a favorable safety profile, the following adverse effects have been reported: syncope, fatigue, nausea, vomiting, abdominal pain, hypotension, anemia, thrombocytopenia, neutropenia, febrile neuropenia, pneumonia, dysuria, and hematuria [116, 117, 120].



*Abbreviations*: MDS, myelodysplastic syndrome; AML, acute myeloid leukemia; OR, overall response; CR, complete response; PR, partial response; HI, hematological improvement; BSC, best supportive care; vs., versus; 20 , secondary; OS, overall survival.

**Table 5.** Decitabine trials in MDS patients.

**Trial Number of patients** 

146 Myelodysplastic Syndromes

Kantarjian et al. Cancer 2006

Ruter et al. Cancer 2006

Kantarjian et al. Cancer 2007

Kantarjian et al. Blood 2007

Kantarjian et al. Cancer 2007

Borthakur et al. Leuk Lymphoma 2008

Steensma et al. JCO 2009

Lubbert et al. JCO 2011

**Phase and design of trial**

randomized multicenter

Decitabine vs.

BSC

Low dose decitabine as salvage therapy at relapse

Prognostic factors associated with outcome

Optimal dosage of decitabine

Decitabine vs. AML type of intensive chemotherapy

Efficacy of decitabine after failure of vidaza

Efficacy & safety of decitabine as outpatient regimen

Decitabine vs.

BSC

170 Phase II

22 Phase II

115 Phase II

95 Phase II

491 Phase II

pooled analysis of 3 trials

1 single center

randomized single center

historical comparison of 2 groups of patients at single center

14 Phase II early results

99 Phase II

233 Phase III- 2 arms multicenter

multicenter non randomized **Study focus Results Total**

OR: 17% CR: 9% PR: 8% HI: 13%

OR: 45% CR: 4.5% PR: 9.1% HI: 31.8%

OR: 70% CR: 35% PR: 2% HI: 10–23%

OR: 73% CR: 34–39% PR: 1% HI: 14–24%

34%

OR: 28% CR: 21% HI: 7%

OR: 32% CR: 17% HI: 18%

CR: 13% PR: 6% HI: 15%

CR: decit: 43% vs. intensive chemotherapy:

**dose per course/ interval between cycles**

135 mg/m2 6 weekly

135 mg/m2 6 weekly

100 mg/m2 4 weekly

100 mg/m2

100 mg/m2

100 mg/m2 4 weekly

100 mg/m2 4 weekly

95 mg/m2 6 weekly


–

**Median number of courses**

3 12.1

≥7 Not

≥6 27% over 18 months

– – 22

3 4 months 6 months

5 – 19.4

months vs. 6.1 months

4 8.8

**Time to AML progress**

months vs. 7.8 months

3 - 37.5

reached

**Median survival**

14 months vs. 14.9 months

months

22 months

19 months

months vs. 12 months

months

PFS: 6.6 months vs. 3 months

#### **11. The role of HSCT in high-risk MDSs including monosomy 7**

#### **11.1. HSCT in adults with MDSs**

Allogeneic HSCT is the only potentially curative therapy for MDS patients [4, 82, 122–124]. Recently, HSCT is being used with increasing frequency in patients with MDS, partly due to the development of novel conditioning therapies, such as nonmyeloablative conditioning, that allow HSCT to be offered to older patients [4, 122]. Also, the use of immunomodulatory drugs and hypomethylating agents prior to HSCT has shown efficacy in (1) controlling disease, that is, a bridging approach to HSCT, and (2) tumor debulking before HSCT [4, 122]. The indications of allogeneic HSCT in adults with MDS are shown in **Table 6** [4, 82, 122–125].

MDS patients with monosomy 7 or complex cytogenetics and preserved BM are considered indications for immediate rather than delayed HSCT. Secondary MDS is another special indication for HSCT [82, 125]. Currently, patients with therapy-related MDS (t-MDS) are being treated using the same paradigm as in *de novo* MDS [82]. Despite its curative potential, the role of allogeneic HSCT in the treatment of elderly patients with MDS is less well defined than in younger individuals [4]. The following issues related to HSCT remain an area of intense investigations: (1) pretransplant disease burden, (2) optimal conditioning therapies, (3) optimal donor selection, (4) optimal stem cell source, (5) GVHD prophylaxis, and (6) post‐ transplant relapse [122]. The following complications of allogeneic HSCT: GVHD, infections, and non-relapse mortality may offset the benefits of allogeneic HSCT over medical therapies [82]. Despite the remarkable improvement in both efficacy and safety of HSCT over the past two decades, therapy-related morbidity and mortality as well as disease relapse still pose significant risks to transplanted patients [82, 122, 123]. Methods employed to prevent and treat relapse of MDS following HSCT include (1) donor lymphocyte infusion (DLI), (2) hypome‐ thylating agents, (3) novel cellular therapies including vaccination, and (4) use of alloreactive natural killer cells [4, 122].


*Abbreviations*: MDS, myelodysplastic syndrome; t-MDS, therapy-related myelodysplastic syndrome; RAEB, refractory anemia with excess of blasts; RAEB-t, refractory anemia with excess of blasts in transformation; SCT, hematopoietic stem cell transplantation.

**Table 6.** Indications for allogeneic HSCT in patients with MDS.

Predictors of outcome of allogeneic HSCT in MDS patients include: (1) disease stage including blast count, (2) transfusion dependence, and (3) karyotype, cytogenetic abnormalities and molecular aberrations or genetic mutations such as: monosomal karyotype, complex cytoge‐ netic and TP53 mutation [82, 122, 125].

#### **11.2. HSCT in children with MDSs**

Allogeneic HSCT is the only potentially curative therapy for children with MDSs, particularly those having JMML [73, 126]. The indications of allogeneic HSCT in children with MDSs are shown in **Table 6** [73, 126–128]. Relapse rate following allogeneic HSCT performed for JMML may reach 50% or more [73]. Children with MDS and JMML should be referred for allogeneic HSCT soon after making the diagnosis in order to prevent disease progression as pretransplant chemotherapy does not appear to improve outcome [127, 128]. Predictors of poor outcome of allogeneic HSCT in children with MDS include (1) monosomy 7, (2) age more than 4 years at transplant, (3) relapse after HSCT, (4) female gender, and (5) human leukocyte antigen (HLA) mismatched allografts [73].

#### **11.3. HSCT in higher risk MDS patients**

two decades, therapy-related morbidity and mortality as well as disease relapse still pose significant risks to transplanted patients [82, 122, 123]. Methods employed to prevent and treat relapse of MDS following HSCT include (1) donor lymphocyte infusion (DLI), (2) hypome‐ thylating agents, (3) novel cellular therapies including vaccination, and (4) use of alloreactive

> (A) Definite indications: 1- Intermediate 2 IPSS 2- High-risk IPSS (B) Probable indications: 1- t-MDS or secondary MDS


cytogenetics

*Abbreviations*: MDS, myelodysplastic syndrome; t-MDS, therapy-related myelodysplastic syndrome; RAEB, refractory anemia with excess of blasts; RAEB-t, refractory anemia with excess of blasts in transformation; SCT, hematopoietic

Predictors of outcome of allogeneic HSCT in MDS patients include: (1) disease stage including blast count, (2) transfusion dependence, and (3) karyotype, cytogenetic abnormalities and molecular aberrations or genetic mutations such as: monosomal karyotype, complex cytoge‐

Allogeneic HSCT is the only potentially curative therapy for children with MDSs, particularly those having JMML [73, 126]. The indications of allogeneic HSCT in children with MDSs are shown in **Table 6** [73, 126–128]. Relapse rate following allogeneic HSCT performed for JMML may reach 50% or more [73]. Children with MDS and JMML should be referred for allogeneic HSCT soon after making the diagnosis in order to prevent disease progression as pretransplant chemotherapy does not appear to improve outcome [127, 128]. Predictors of poor outcome of allogeneic HSCT in children with MDS include (1) monosomy 7, (2) age more than 4 years at transplant, (3) relapse after HSCT, (4) female gender, and (5) human leukocyte antigen (HLA)-

2- Packed Red blood cell transfusions refractory to:

4- At least one line of cytopenia with multilineage dysplasia 5- High risk chromosomal abnormalities: monosomy 7 and complex

3- Severe neutropenia or thrombocytopenia

6- High percentage of blasts [≥10%]

natural killer cells [4, 122].

148 Myelodysplastic Syndromes

transformation (RAEB-t)

a- transfusion dependence b- cytogenetic abnormalities

stem cell transplantation.

(RAEB)

(t-MDS)

1- Refractory anemia with excess of blasts

2- Refractory anemia with excess of blasts in

3- Chemotherapy or radiotherapy related MDS

4- Juvenile myelomonocytic leukemia 5- Refractory cytopenias associated with:

**In children In adults**

**Table 6.** Indications for allogeneic HSCT in patients with MDS.

netic and TP53 mutation [82, 122, 125].

**11.2. HSCT in children with MDSs**

mismatched allografts [73].

Patients with higher risk MDS who have an HLA-matched donor should be transplanted early before progression of their disease or acquisition of a nonhematological contraindication to HSCT [129]. In patients with intermediate-2 or high-risk MDS, aged 60–79 years, subjected to reduced intensity conditioning allogeneic HSCT, life expectancy is about 36 months compared to 28 months in patients not subjected to HSCT, that is, HSCT in this group of patients has a survival advantage [130]. Patients with higher risk MDS should be treated with either hypomethylating agents or HSCT [131]. It is justified to offer patients with higher risk MDS who have an HLA identical donor an allograft [129]. Retrospective studies have concluded that patients with higher risk MDS have a survival advantage over demethylating agents if they can be offered an early allogeneic HSCT [129]. Transplant-related mortality remains high in HSCT, it ranges between 10% and 40% particularly after myeloablative conditioning therapy in elderly individuals [129]. Long-term survival ranges between 30% and 60% depending on patient characteristics, disease risk, type of donor, source of stem cells, and complications that evolve following HSCT [129].

#### **12. Conclusions and future directions**

In children and adults, high-risk MDSs including monosomy 7 are often complicated by various degrees of BM suppression, infectious complications, severe iron overload, and transformation into AML. Management of these disorders includes (1) supportive care that comprises transfusion of blood products, antimicrobials, and iron chelation therapy, (2) epigenetic therapies including histone deacetylase inhibitors and hypomethylating agents such as azacytidine and decitabine, and (3) various types of allogeneic HSCT. Recently, the role of allogeneic HSCT in high-risk MDSs is increasing due to the introduction of the new conditioning therapies that have allowed the application of this curative modality of therapy not only to older patients, but also to individuals with medical comorbidities. However, in patients with familial causes of their MDSs of BM failure planned for allogeneic HSCT using a sibling donor, great caution should be exercised and enough investigations should be performed before clearing the sibling for donation. The role of certain growth factors in the management of patients with high-risk MDS is controversial as G-CSF has been reported to accelerate the progression into acute leukemia.

The recent developments in the diagnostics of MDSs and the recently introduced therapeutic agents such as rigosertib, clofarabine, sapacitabine, and BCL2 inhibitors as well as the evolving modalities of HSCT are likely to improve the outcome of patients with higher risk MDSs significantly.

#### **Author details**

Khalid Ahmed Al-Anazi

Address all correspondence to: kaa\_alanazi@yahoo.com

Department of Adult Hematology and Hematopoietic Stem Cell Transplantation, Oncology Center, King Fahad Specialist Hospital, Dammam, Saudi Arabia

#### **References**


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2012 Apr 13.

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Address all correspondence to: kaa\_alanazi@yahoo.com

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Center, King Fahad Specialist Hospital, Dammam, Saudi Arabia

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#### **Chapter 6**

## **Chronic Myelomonocytic Leukaemia**

#### Andreas Himmelmann

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63193

#### **Abstract**

The classification, pathobiology and clinical management of chronic myelomonocytic leukaemia (CMML) are reviewed. Three important issues are identified: (1) CMML should be recognised as a unique clinical entity and as distinct from myelodysplastic syn‐ dromes (MDSs). Somatic mutations of a restricted set of genes are frequent in CMML. (2) Risk stratification for CMML patients should utilise new CMML‐specific prognostic scoring systems. (3) Until randomised clinical trials have defined the role of new drugs (especially of the hypomethylating agents), treatment must focus on the main symp‐ toms and aim at quality‐of‐life improvement.

**Keywords:** chronic myelomonocytic leukaemia, myelodysplastic/myeloproliferative syndrome, somatic mutations, prognosis, therapy

#### **1. Introduction**

Chronic myelomonocytic leukaemia (CMML) is a rare haematological neoplasm character‐ ised by a persistent peripheral blood monocytosis and both myelodysplastic and myeloproli‐ ferative features. Our understanding of this disease has undergone profound changes in recent years. Initially it was classified as a subtype of myelodysplastic syndromes (MDSs), and it is now recognised as a unique disease. This reclassification has been substantiated by recent advances in the genetic and molecular pathogenesis of CMML, which have confirmed that CMML is biologically distinct from MDS with a different pattern of somatic mutations and a different molecular ontogeny [1]. In addition, CMML‐specific prognostic scoring systems (CPSSs) have been established by various groups. These differ from those commonly used in MDS and, for the first time, also include molecular markers. Hopefully, these efforts will culminate in CMML‐specific treatments in the near future. The following chapter will summa‐

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

rise these developments starting with a discussion of CMML classification and ending with an outlook on new treatment approaches.

#### **2. Diagnosis and classification**

The first reports comprising significant numbers of patients with CMML were published around 40 years ago [2, 3]. These early series already noted a considerable clinical diversity, describing myelodysplasia and cytopenia accompanied by leukocytosis and other myelopro‐ liferative symptoms such as splenomegaly. Nevertheless, the first French–American–British (FAB) Cooperative Group classification of MDS in 1982 included CMML as a MDS subtype, emphasising its dysplastic features but not its diversity [4]. The diagnostic criteria proposed included more than 1 × 10<sup>9</sup> /L blood monocytes, bone marrow blasts of 20% or less, peripheral blasts <5%, and bone marrow dysplastic features in at least one haematopoietic lineage (**Table 1**). To account for the clinical diversity mentioned earlier, the FAB group later intro‐ duced a subclassification by dividing patients into two groups based on the leukocyte count at diagnosis [5]. Patients with a leukocyte count above 13 × 10<sup>9</sup> /L were considered to have a myeloproliferative form (MPD‐CMML), those with a leukocyte count below 13 × 10<sup>9</sup> /L a myelodysplastic form (MDS‐CMML). This arbitrary threshold has been controversial, and its clinical relevance was tested in several cohorts of patients with CMML. In a retrospective analysis of 158 patients (81 patients with MDS‐CMML and 77 patients with MPD‐CMML), Germing et al. found no significant difference in 2‐year overall survival (OS) between these subgroups. The likelihood of transformation to AML was higher in the MDS‐CMML group, but this difference was not statistically significant [6]. Voglova et al. [7] described the frequent transition of MDS‐CMML to MPD‐CMML, suggesting that the two subgroups might represent different stages of the same disease rather than two different entities. Onida et al. analysed a cohort of 213 patients with CMML (35% with MDS‐CMML and 65% with MPD‐CMML) and could also not find a statistically significant difference in survival after 12 months. There was, however, a trend for a better survival of patients with MPD‐CMML after 16 months [8]. To resolve these diagnostic inconsistencies, the WHO (2001) classification of myeloid neoplasms defined a new group of overlap syndromes called myelodysplastic/myeloproliferative diseases (MDS/MPS). Besides CMML, atypical (bcr‐abl negative) CML (aCML), juvenile myelomonocytic leukaemia (JMML) and myelodysplastic/myeloproliferative disease, unclas‐ sifiable (MDS/MPS‐U) were placed in this group [9]. The details of the diagnostic criteria for CMML are listed in **Table 1**. Further, two CMML subtypes were recognised: CMML‐1 with PB blast <5% and <10% BM blasts and CMML‐2 with 5–19% PB blasts and 10–19% BM blasts. The prognostic significance of these subgroups was confirmed in a reanalysis of the Düsseldorf registry of 300 CMML patients. The 5‐year risk of transformation to AML was 63% for patients with CMML‐2 compared to only 18% for patients with CMML‐1 [10]. More recently, the same group found that further subclassification of CMML based on medullary blast count could provide additional prognostic information [11]. Patients with the new subtype of CMML‐0 (defined as <5% medullary blasts) had a better prognosis than those with CMML‐1 (OS 31 vs. 19 months). These results have not yet been reproduced in an independent cohort.

The 2008 revision of the WHO classification has maintained the main diagnostic criteria for CMML, with one additional feature [12]. In particular, the recognition of an associated eosinophilia is an important clue to an underlying rearrangement of the platelet‐derived growth factor receptor, alpha or beta polypeptide (*PDGFRA/B*) gene. If this molecular lesion is found, the case should be classified as a myeloid neoplasm with eosinophilia associated with *PDGFRA/B* rearrangement. These patients often respond exquisitely to imatinib [13].

Diagnostic difficulties most commonly arise in the distinction between CMML and aCML, because a monocytosis can be present in the latter. In difficult cases, the pattern of somatic mutations might be exploited in the future [14, 15]. For example, the co‐existence of a serine/ arginine‐rich splicing factor 2 (*SRSF2*) and a ten–eleven translocation‐2 (*TET2*) mutation suggests CMML, a SET nuclear proto‐oncogene binding protein1 (*SETBP1*) mutation points towards aCML.

The presence of a peripheral blood monocytosis is a diagnostic prerequisite for the diagnosis of CMML but can also be caused by a number of reactive conditions. In patients with clinical or laboratory signs of inflammation, such as fever, arthritis, increased C‐reactive protein or elevated erythrocyte sedimentation rate, the diagnosis of CMML should be made with caution. Often, re‐evaluation is necessary once the signs of inflammation have subsided. A recent study suggests that the distinction between reactive monocytosis and CMML might be possible by immunophenotyping, but this finding needs further confirmation in independent studies [16].


FAB, French‐American‐British; PB, peripheral blood; BM, bone marrow.

**Table 1.** Comparison of the FAB and WHO classifications of CMML.

#### **3. Epidemiology and clinical features**

rise these developments starting with a discussion of CMML classification and ending with an

The first reports comprising significant numbers of patients with CMML were published around 40 years ago [2, 3]. These early series already noted a considerable clinical diversity, describing myelodysplasia and cytopenia accompanied by leukocytosis and other myelopro‐ liferative symptoms such as splenomegaly. Nevertheless, the first French–American–British (FAB) Cooperative Group classification of MDS in 1982 included CMML as a MDS subtype, emphasising its dysplastic features but not its diversity [4]. The diagnostic criteria proposed

blasts <5%, and bone marrow dysplastic features in at least one haematopoietic lineage (**Table 1**). To account for the clinical diversity mentioned earlier, the FAB group later intro‐ duced a subclassification by dividing patients into two groups based on the leukocyte count

myeloproliferative form (MPD‐CMML), those with a leukocyte count below 13 × 10<sup>9</sup>

19 months). These results have not yet been reproduced in an independent cohort.

myelodysplastic form (MDS‐CMML). This arbitrary threshold has been controversial, and its clinical relevance was tested in several cohorts of patients with CMML. In a retrospective analysis of 158 patients (81 patients with MDS‐CMML and 77 patients with MPD‐CMML), Germing et al. found no significant difference in 2‐year overall survival (OS) between these subgroups. The likelihood of transformation to AML was higher in the MDS‐CMML group, but this difference was not statistically significant [6]. Voglova et al. [7] described the frequent transition of MDS‐CMML to MPD‐CMML, suggesting that the two subgroups might represent different stages of the same disease rather than two different entities. Onida et al. analysed a cohort of 213 patients with CMML (35% with MDS‐CMML and 65% with MPD‐CMML) and could also not find a statistically significant difference in survival after 12 months. There was, however, a trend for a better survival of patients with MPD‐CMML after 16 months [8]. To resolve these diagnostic inconsistencies, the WHO (2001) classification of myeloid neoplasms defined a new group of overlap syndromes called myelodysplastic/myeloproliferative diseases (MDS/MPS). Besides CMML, atypical (bcr‐abl negative) CML (aCML), juvenile myelomonocytic leukaemia (JMML) and myelodysplastic/myeloproliferative disease, unclas‐ sifiable (MDS/MPS‐U) were placed in this group [9]. The details of the diagnostic criteria for CMML are listed in **Table 1**. Further, two CMML subtypes were recognised: CMML‐1 with PB blast <5% and <10% BM blasts and CMML‐2 with 5–19% PB blasts and 10–19% BM blasts. The prognostic significance of these subgroups was confirmed in a reanalysis of the Düsseldorf registry of 300 CMML patients. The 5‐year risk of transformation to AML was 63% for patients with CMML‐2 compared to only 18% for patients with CMML‐1 [10]. More recently, the same group found that further subclassification of CMML based on medullary blast count could provide additional prognostic information [11]. Patients with the new subtype of CMML‐0 (defined as <5% medullary blasts) had a better prognosis than those with CMML‐1 (OS 31 vs.

at diagnosis [5]. Patients with a leukocyte count above 13 × 10<sup>9</sup>

/L blood monocytes, bone marrow blasts of 20% or less, peripheral

/L were considered to have a

/L a

outlook on new treatment approaches.

164 Myelodysplastic Syndromes

**2. Diagnosis and classification**

included more than 1 × 10<sup>9</sup>

CMML is a rare disease of the elderly. Two recent population‐based studies found a similar age standardised annual incidence rate of approximately 0.3–0.4/100,000/year [17, 18]. Median age was 70–75 years, and there was a slight male predominance. The study from the Nether‐ lands found that the diagnosis was made in a non‐university setting in 78%, indicating that many patients are managed by practising haematologists. This observation emphasises the need to perform clinical studies also in a community‐based setting to include as many patients as possible. The 5‐year relative survival was poor (16–20%) in this study and did not improve over time. Therapy‐related CMML (t‐CMML), defined as occurring after chemotherapy, radiotherapy or both, is considered rare but was found in 10% in a recent MD Anderson Cancer Centre (MDACC) series. Patients with t‐CMML had a significantly worse median OS com‐ pared to patients with de novo disease (13 vs. 20 months), most likely due to a higher rate of intermediate‐ or high‐risk cytogenetic abnormalities [19]. Similar results were reported in a series from the Mayo Clinic (median OS 11 vs. 20 months) [20].

#### **3.1. Clinical features**

Monocytosis can be an incidental finding in an otherwise asymptomatic patient. In other cases, it is accompanied by anaemia (in about 50% of patients at diagnosis) and/or thrombocytopenia. Frequently, a haematological prodrome, for example an unclear thrombocytopenia, can be observed. In about 30–60% of patients, leukocyte counts >13 × 109 /L are found, often with clinical signs of myeloproliferation such as splenomegaly in around 30% [8]. Hepatospleno‐ megaly is more frequent (25–50% of patients) in the myeloproliferative variant [21]. Many patients experience constitutional symptoms, fatigue, night sweats and occasionally bone pain.

In contrast to MDS, involvement of various organ systems has been described in CMML patients. Skin lesions can be an indicator of leukemic transformation [22]. Serosal infiltration causing pleural or pericardial effusion is quite frequent and can be difficult to treat [23]. Local instillation of mitoxantrone, in combination with systemic chemotherapy, has been used with some success in such cases. A case of widespread gastric involvement mimicking metastatic gastric carcinoma was recently seen in our practice (own unpublished observation). Cases of uncontrollable haematuria caused by CMML involvement of the urogenital tract have also been described [24, 25].

An association with autoimmune‐mediated disorders is frequently seen in CMML. For example, in a study of 123 CMML patients, 20% had at least one associated disorder, most commonly immune thrombocytopenia (ITP), gout or psoriasis [26]. Importantly, ITP seems to respond well to standard treatment used in primary ITP, such as steroids and splenectomy [27].

Over time transformation into acute myeloid leukaemia occurs in approximately 30% of patients. The rate of transformation varies according to the risk profile at diagnosis. A sudden rise in the leukocyte count does not necessarily indicate leukemic transformation but can be an expression of increased myeloproliferation. A careful evaluation of the blast count is important in this situation.

#### **3.2. Laboratory and pathologic findings**

In the peripheral blood, monocytes can be normal or display atypical features such as fine nuclear chromatin or abnormal nuclear lobulations. In the myeloproliferative variant, median absolute monocyte counts ranging from 4.2 × 10<sup>9</sup> to 7.7 × 10<sup>9</sup> /L have been reported [21]; in general, the median monocyte count is around 2 × 10<sup>9</sup> –3 × 10<sup>9</sup> /L. Morphologic evidence of dysgranulopoesis is often seen in CMML, while dysmegakaryopoiesis and dyserythropoiesis are less frequent. The bone marrow is usually hypercellular with an elevated myelopoiesis‐to‐ erythropoiesis ratio. By definition, the blast count is <20%. When enumerating blasts, mono‐ blasts and promonocytes should be included. A helpful morphological classification of monocytic precursors, particularly defining promonocytes in CMML, is available [28]. Monocytic precursors including monoblasts are frequently CD34 negative. Therefore, there is a risk of underestimating the blast number by relying only on CD34 staining in bone marrow biopsies or flow cytometry. The medullary blast count should be determined in good quality bone marrow aspirates that have also been stained for esterase.

#### **4. Cytogenetics**

age was 70–75 years, and there was a slight male predominance. The study from the Nether‐ lands found that the diagnosis was made in a non‐university setting in 78%, indicating that many patients are managed by practising haematologists. This observation emphasises the need to perform clinical studies also in a community‐based setting to include as many patients as possible. The 5‐year relative survival was poor (16–20%) in this study and did not improve over time. Therapy‐related CMML (t‐CMML), defined as occurring after chemotherapy, radiotherapy or both, is considered rare but was found in 10% in a recent MD Anderson Cancer Centre (MDACC) series. Patients with t‐CMML had a significantly worse median OS com‐ pared to patients with de novo disease (13 vs. 20 months), most likely due to a higher rate of intermediate‐ or high‐risk cytogenetic abnormalities [19]. Similar results were reported in a

Monocytosis can be an incidental finding in an otherwise asymptomatic patient. In other cases, it is accompanied by anaemia (in about 50% of patients at diagnosis) and/or thrombocytopenia. Frequently, a haematological prodrome, for example an unclear thrombocytopenia, can be

clinical signs of myeloproliferation such as splenomegaly in around 30% [8]. Hepatospleno‐ megaly is more frequent (25–50% of patients) in the myeloproliferative variant [21]. Many patients experience constitutional symptoms, fatigue, night sweats and occasionally bone pain.

In contrast to MDS, involvement of various organ systems has been described in CMML patients. Skin lesions can be an indicator of leukemic transformation [22]. Serosal infiltration causing pleural or pericardial effusion is quite frequent and can be difficult to treat [23]. Local instillation of mitoxantrone, in combination with systemic chemotherapy, has been used with some success in such cases. A case of widespread gastric involvement mimicking metastatic gastric carcinoma was recently seen in our practice (own unpublished observation). Cases of uncontrollable haematuria caused by CMML involvement of the urogenital tract have also

An association with autoimmune‐mediated disorders is frequently seen in CMML. For example, in a study of 123 CMML patients, 20% had at least one associated disorder, most commonly immune thrombocytopenia (ITP), gout or psoriasis [26]. Importantly, ITP seems to respond well to standard treatment used in primary ITP, such as steroids and splenectomy [27].

Over time transformation into acute myeloid leukaemia occurs in approximately 30% of patients. The rate of transformation varies according to the risk profile at diagnosis. A sudden rise in the leukocyte count does not necessarily indicate leukemic transformation but can be an expression of increased myeloproliferation. A careful evaluation of the blast count is

In the peripheral blood, monocytes can be normal or display atypical features such as fine nuclear chromatin or abnormal nuclear lobulations. In the myeloproliferative variant, median

/L are found, often with

series from the Mayo Clinic (median OS 11 vs. 20 months) [20].

observed. In about 30–60% of patients, leukocyte counts >13 × 109

**3.1. Clinical features**

166 Myelodysplastic Syndromes

been described [24, 25].

important in this situation.

**3.2. Laboratory and pathologic findings**

Clonal chromosomal abnormalities occur in approximately 30% of patients with CMML [29, 30]. The most frequent abnormalities are +8 (20–25%), -Y (20%), monosomy 7 and deletion 7q (14%), deletion 20q (8%). A complex karyotype was found in 11%. In contrast to MDS, del5q is very rare. Patients with an abnormal karyotype tended to be older, more anaemic and had a higher peripheral blood and bone marrow blast count [30]. Additional sex combs like 1 (*ASXL1*) mutations were associated with an abnormal karyotype, *SRSF2* mutations with a normal karyotype [31]. Cytogenetic abnormalities were also found to be of prognostic relevance (see Section 6).

#### **5. Molecular findings**

Large‐scale sequencing studies in myeloid malignancies have lead to important insights into disease biology. These studies have shown one of the highest rates of acquired somatic mutations in CMML patients. For example, in a study by Meggendorfer et al. [31], at least one mutation in 9 recurrently mutated genes was found in 93% of 275 CMML patients. This study and several others also identified clear differences in frequency of mutations in key cellular pathways between CMML and MDS. In addition, the genomic landscape in CMML demon‐ strates a much smaller molecular heterogeneity compared to a more diffuse mutation profile in MDS [32].

**Table 2** summarises the frequency of mutations sorted by the cellular pathway affected [33]. In contrast to MDS, genes serving in different signalling pathways are frequently mutated in CMML. As a biological correlate, an increased sensitivity to GM‐CSF has been found in vitro. It appears to be mediated by the STAT pathway, since inhibition of proliferation was observed by the JAK2 inhibitor ruxolitinib [34], indicating the therapeutic potential of these agents or anti GM‐CSF monoclonal antibodies.


**Table 2.** Frequency of somatic mutations in CMML.

Significant differences were also found in the frequency of mutations affecting RNA splicing. While mutations in *SRSF2* are very common in CMML (40–45%), they are found in <10% of MDS patients, with an enrichment in subtypes with blast excess [31, 32]. Similarly, mutations in epigenetic regulators, among them *TET2* and *ASXL1*, occur much more frequently in CMML than in MDS. For example, using next generation sequencing, *TET2* mutations were found in only 39 of 320 patients with MDS (12%) but in 16 of 35 patients with CMML (46%) [35].

TET2 belongs to the ten–eleven translocations family of proteins and participates in the conversion of 5‐methylcytosine to 5‐hydroxymethylcytosine. TET2 function depends on the presence of alpha‐ketoglutarate, which is produced by isocitrate dehydrogenase 1 and 2 (IDH1/2). *TET2* and *IDH1/2* mutations are mutually exclusive and lead to promoter *hype*rmethylation [36], providing a potential explanation for the mode of action of the hypo‐ methylating agents (HMAs). *TET2* mutations appear to be particularly important for CMML pathophysiology and development of its characteristic phenotype. In a series of elegant experiments with samples from CMML patients, Itzykson et al. [37] have shown that early clonal dominance, particularly in a *TET2*‐mutated clone, promotes granulomonocytic differ‐ entiation. Knockdown experiments of *TET2* in human cord blood CD34+ cells have also found perturbation of myeloid development with promotion of the granulomonocytic lineage [38]. Furthermore, the early occurrence of *TET2* mutations could predetermine the acquisition of secondary mutations, for example in *SRSF2*, leading to characteristic mutational combinations. Besides its role in understanding disease biology and classification, mutational analysis is likely to impact on various clinical aspects, for example prognostication (discussed below) and prediction of treatment response.

#### **6. Prognosis**

**Cellular pathway Gene Frequency (%)**

*NRAS* 11% *CBL* 10% *JAK2* 10% *SETBP1* 10%

*ZRR2* 10% *U2AF35* 10%

*ASXL1* 50% *EZH2* 8%

Significant differences were also found in the frequency of mutations affecting RNA splicing. While mutations in *SRSF2* are very common in CMML (40–45%), they are found in <10% of MDS patients, with an enrichment in subtypes with blast excess [31, 32]. Similarly, mutations in epigenetic regulators, among them *TET2* and *ASXL1*, occur much more frequently in CMML than in MDS. For example, using next generation sequencing, *TET2* mutations were found in only 39 of 320 patients with MDS (12%) but in 16 of 35 patients with CMML (46%) [35].

TET2 belongs to the ten–eleven translocations family of proteins and participates in the conversion of 5‐methylcytosine to 5‐hydroxymethylcytosine. TET2 function depends on the presence of alpha‐ketoglutarate, which is produced by isocitrate dehydrogenase 1 and 2 (IDH1/2). *TET2* and *IDH1/2* mutations are mutually exclusive and lead to promoter *hype*rmethylation [36], providing a potential explanation for the mode of action of the hypo‐ methylating agents (HMAs). *TET2* mutations appear to be particularly important for CMML pathophysiology and development of its characteristic phenotype. In a series of elegant experiments with samples from CMML patients, Itzykson et al. [37] have shown that early clonal dominance, particularly in a *TET2*‐mutated clone, promotes granulomonocytic differ‐ entiation. Knockdown experiments of *TET2* in human cord blood CD34+ cells have also found perturbation of myeloid development with promotion of the granulomonocytic lineage [38]. Furthermore, the early occurrence of *TET2* mutations could predetermine the acquisition of secondary mutations, for example in *SRSF2*, leading to characteristic mutational combinations. Besides its role in understanding disease biology and classification, mutational analysis is

**Signalling** *KRAS* 8%

**RNA splicing** *SRSF2* 46%

**Epigenetic regulation** *TET2* 58%

**Transcription** RUNX1 15%

Numbers based on Refs. [1, 33].

168 Myelodysplastic Syndromes

**Table 2.** Frequency of somatic mutations in CMML.

#### **6.1. Individual parameters**

The importance of the medullary blast count as a prognostic variable was discussed before and forms the basis of the subclassification of CMML into CMML‐1 and CMML‐2. The prognostic significance of cytogenetic abnormalities was first described by the Spanish MDS group in a cohort of 414 CMML patients. Three risk cytogenetic categories were identified in that study: low risk (normal karyotype and loss of chromosome Y as single abnormality), intermediate (all other single or double abnormalities) and high risk (trisomy 8, abnormalities of chromosome 7 and complex karyotype). The OS at 5 years for these risk groups was 35, 24 and 4%, respectively [29]. In a recent large collaborative analysis of 409 patients with CMML, slightly different cytogenetic risk groups were defined: low risk [normal, sole -Y, sole der(3q)], intermediate (all karyotypes not belonging to high or low risk group) and high risk (complex and monosomal karyotype). In contrast to the Spanish study, trisomy 8 was placed in the intermediate risk group. The median OS was 41, 20 and 3 months, respectively [30]. The mutational status of several genes has also shown to be of prognostic relevance, although conflicting results were found in different cohorts. For example, mutations in *TET2* were associated with no effect on outcome in one study [35] and with an adverse outcome in another study [39]. In a large series of 312 patients tested for a number of genes, only *ASXL1* mutations had a negative prognostic value in multivariate analysis [40]. This finding was confirmed in a larger cohort of 466 patients (including the 312 patients from the original series) [41]. In another large study of 275 CMML patients, no effect of *SRSF2* mutations on survival was observed [31]. In summary, *ASXL1* mutational status has emerged as a robust prognostic variable and has thus been incorporated into CPSSs, as discussed below.

#### **6.2. Prognostic scoring systems**

A number of prognostic scoring systems for CMML patients have been developed in the past, none of which however was universally used [42]. This is in contrast to MDS where the International Prognostic Scoring System (IPSS) and its recently revised version (IPSS‐R) form the basis of treatment decisions in studies as well as in clinical practice. More recently, novel CMML‐specific scoring systems have been described that incorporate cytogenetic or molecular information. These scores appear to be more precise and will be discussed further. First, the CPSS has been developed by the Spanish MDS group in a cohort of 558 patients [43]. It uses WHO/FAB subtype, a CMML‐specific cytogenetic risk categorisation and transfusion depend‐ ence to divide patients into four risk groups (low, intermediate‐1 or intermediate‐2, and high) with a median OS of 72, 31, 13 and 5 months, respectively). This model demonstrated for the first time that cytogenetic abnormalities are of prognostic relevance in CMML. Notably, it highlights that the significance of individual cytogenetic abnormalities can vary between CMML and MDS. For example, trisomy 8 carries an adverse prognosis in CMML but not in MDS. The CPPS has been externally validated in a cohort of 274 patients. Second, the French cooperative MDS group (GFM) developed a prognostic model that included the presence of *ASXL1* mutations, age (>65 years being an adverse prognostic factor), and haematological parameters (WBC > 15 × 10<sup>9</sup> /L, platelet count <100 × 10<sup>9</sup> /L and haemoglobin <11 g/dl being adverse factors). It stratifies patients into 3 risk categories with a median OS of not reached, 38.5 and 14.4 months, respectively, and has also been externally validated [40]. Lastly, the group from the Mayo Clinic has improved on their original prognostic model by incorporating *ASXL1* mutational status. This new score has been termed Mayo Molecular Model (MMM) and was developed in corporation with the GFM [41]. Five risk factors affected median survival in multivariate analysis*: ASXL1* mutation status, absolute monocyte count >10 × 10<sup>9</sup> /L, haemoglobin levels <10 g/dl, platelet count <100 × 10<sup>9</sup> /L, and circulating immature myeloid cells. It divides patients into four risk categories with median survival of 97, 59, 37 and 16  months, respectively. Importantly, this new score can identify low risk patients (by the original Mayo Clinic score) with a high risk of progression (without or with *ASXL1* mutation: median survival 99 vs. 44 months).

Particularly when considering allogeneic stem cell transplantation, prognostication in younger CMML patients, defined as younger than 65 years, is crucial. A retrospective analysis of 261 such patients has identified several adverse prognostic factors that differ from those in the general CMML population. In addition to anaemia and *ASXL1* mutations, an increased circulating blast count, *SRSF2* mutations and the cytogenetic risk classification of the Mayo‐ French consortium were independently prognostic. In this study, *ASXL1* and *SRSF2* mutation status did not influence response to HMAs or transplantation outcome [44].

In summary, it is evident that CPSSs that include cytogenetic and/or molecular parameters should be employed in the future.

#### **7. Therapy**

#### **7.1. General considerations**

Although providing little prognostic information, the concept of a myelodysplastic and a myeloproliferative variant of CMML is helpful in guiding treatment. In particular, patients suffering mainly from uncontrolled myeloproliferation may require rapidly acting cytoreductive treatment. On the other hand, patients with symptoms due to marrow failure require treatment aiming at restoring adequate peripheral blood counts. No treatment so far has been shown to prolong survival or to alter the natural history of the disease. However, randomised studies in CMML patients have not yet been performed, with one exception [45]. Typically, CMML patients (excluding those with MPD‐CMML) were included in MDS trials, albeit in small numbers precluding a meaningful statistical analysis. For example, in the AZA‐ 001 trial that led to the registration of azacitidine in higher risk MDS only 16 of the 358 enrolled patients had CMML [46].

For the treatment of lower risk patients with symptomatic anaemia, erythropoiesis‐stimulating agents (ESA) might be helpful. A 64% erythroid response rate and transfusion independence in 33% of patients was recently reported in a retrospective analysis of 94 CMML patients. Low/ intermediate‐1 CPSS and low endogenous erythropoietin levels were predictors of response [47]. ESA's should be used with caution in patients with myeloproliferative CMML because of the risk of splenic enlargement or rupture.

Younger patients with high‐risk features (for example intermediate‐2 or high risk in the MMM) should be evaluated for eligibility for allogeneic stem cell transplantation. Whether high‐risk patients benefit from early treatment with HMAs, as in high‐risk MDS, must be tested in prospective randomised trials.

#### **7.2. Stem cell transplantation**

CMML and MDS. For example, trisomy 8 carries an adverse prognosis in CMML but not in MDS. The CPPS has been externally validated in a cohort of 274 patients. Second, the French cooperative MDS group (GFM) developed a prognostic model that included the presence of *ASXL1* mutations, age (>65 years being an adverse prognostic factor), and haematological

adverse factors). It stratifies patients into 3 risk categories with a median OS of not reached, 38.5 and 14.4 months, respectively, and has also been externally validated [40]. Lastly, the group from the Mayo Clinic has improved on their original prognostic model by incorporating *ASXL1* mutational status. This new score has been termed Mayo Molecular Model (MMM) and was developed in corporation with the GFM [41]. Five risk factors affected median survival in multivariate analysis*: ASXL1* mutation status, absolute monocyte count >10 × 10<sup>9</sup>

cells. It divides patients into four risk categories with median survival of 97, 59, 37 and 16  months, respectively. Importantly, this new score can identify low risk patients (by the original Mayo Clinic score) with a high risk of progression (without or with *ASXL1* mutation: median

Particularly when considering allogeneic stem cell transplantation, prognostication in younger CMML patients, defined as younger than 65 years, is crucial. A retrospective analysis of 261 such patients has identified several adverse prognostic factors that differ from those in the general CMML population. In addition to anaemia and *ASXL1* mutations, an increased circulating blast count, *SRSF2* mutations and the cytogenetic risk classification of the Mayo‐ French consortium were independently prognostic. In this study, *ASXL1* and *SRSF2* mutation

In summary, it is evident that CPSSs that include cytogenetic and/or molecular parameters

Although providing little prognostic information, the concept of a myelodysplastic and a myeloproliferative variant of CMML is helpful in guiding treatment. In particular, patients suffering mainly from uncontrolled myeloproliferation may require rapidly acting cytoreductive treatment. On the other hand, patients with symptoms due to marrow failure require treatment aiming at restoring adequate peripheral blood counts. No treatment so far has been shown to prolong survival or to alter the natural history of the disease. However, randomised studies in CMML patients have not yet been performed, with one exception [45]. Typically, CMML patients (excluding those with MPD‐CMML) were included in MDS trials, albeit in small numbers precluding a meaningful statistical analysis. For example, in the AZA‐ 001 trial that led to the registration of azacitidine in higher risk MDS only 16 of the 358 enrolled

status did not influence response to HMAs or transplantation outcome [44].

/L and haemoglobin <11 g/dl being

/L, and circulating immature myeloid

/L,

/L, platelet count <100 × 10<sup>9</sup>

parameters (WBC > 15 × 10<sup>9</sup>

170 Myelodysplastic Syndromes

survival 99 vs. 44 months).

should be employed in the future.

**7.1. General considerations**

patients had CMML [46].

**7. Therapy**

haemoglobin levels <10 g/dl, platelet count <100 × 10<sup>9</sup>

Allogeneic stem cell transplant (allo‐SCT) still remains the only curative option for patients with CMML and should be considered in younger patients with high‐risk disease. So far all reports on allo‐SCT in CMML have been retrospective and many included patients with CMML as well as MDS. CMML-specific patient series have only been recently reported, with the EBMT series comprising 513 patients being by far the largest [48]. The median age was 53 years, clearly younger than the median age of CMML patients in general. The non‐relapse mortality at 1 and 4 years was 31 and 41%, respectively. The incidence of relapse at 4 years was 32%, resulting in an estimated 4‐year relapse free survival of 27% and OS of 33%, respectively. Of note, no influence of procedure‐related parameters such as stem cell source, type of donor or T‐cell depletion on outcome was found. Importantly, the only significant parameter associated with an improved outcome was the presence of a complete remission at the time of transplantation. A similar trend was also found in a smaller study [49]. Thus, allo‐SCT can provide long‐term remissions in about 30% of younger patients. The procedure should be performed after achievement of the best possible remission status, either with combination chemotherapy or with HMAs. Although the best preparatory regimen is not known, a recent retrospective study of 83 patients from the MDACC supports the use of HMA before allo‐SCT. The study found a significantly lower incidence of relapse at 3 years post transplant in patients treated with HMA, compared to patients treated with other agents (22 vs. 35%, p = 0.03), resulting in a better 3‐year progression‐free survival [50]. A selection bias might confound these interesting results, since patients who do not progress while treated with HMA (median of 6 cycles in the study) are likely to have a less aggressive disease.

#### **7.3. Cytoreductive treatment**

In patients with symptoms mainly caused by myeloproliferation, cytoreductive treatment is indicated. Several studies have demonstrated the effect of the topoisomerase‐I inhibitor topotecan in patients with CMML. As a single‐agent, complete response rates of up to 28% have been described [51]. Similarly, clinically meaningful responses, including improvement of life threatening pericardial and pleural effusions, as well as cytopenias were reported for the topoisomerase‐II inhibitor etoposide [52]. However, in a randomised study of 105 patients from 43 European centres, hydroxyurea was found to be superior to etoposide in terms of the response rate (60 vs. 36%) and OS (20 vs. 9 months) and has thus remained the treatment of choice for palliative cytoreduction in CMML patients [45]. The experience with intensive chemotherapy in CMML has been disappointing [53]. AML‐like induction therapy is only rarely used, usually as a preparatory regimen before allogeneic stem cell transplantation in patients with an aggressive disease.

#### **7.4. Hypomethylating agents**

The introduction of the HMAs azacitidine and decitabine is likely to transform clinical management of CMML. A growing number of studies have shown considerable single agent activity with very low toxicity. In the largest study so far (76 patients from France, Cleveland Clinic and Lee Moffitt Cancer Center), a response rate of 43%, with 17% complete remissions, was found [54]. Of note, 46% had a proliferative form of CMML, as defined by a leukocyte count of >13 × 10<sup>9</sup> /L. In that study, the presence of more than 10% bone marrow blasts and palpable splenomegaly had a negative impact on survival. A smaller Italian retrospective study analysed the response in 31 patients with CMML (42% with CMML‐1, 58% with CMML‐ 2) who were treated with azacitidine at a dose of 75 or 50 mg/m<sup>2</sup> for 7 days. The overall response rate was 51%, including 45% achieving complete remission [55]. A study from the Austrian Azacitidine registry reported on the outcome of 48 patients treated with azacitidine at 11 different centres [56]. Mean age was 71 years; 40% had CMML‐1; 60% had CMML‐2; and splenomegaly was found in 48%. Even in this unselected cohort with several high‐risk features, there was a surprising response rate of 70%, including 22% complete responses. Matched paired analysis suggested a better 2‐year‐survival when compared to best supportive care (62 vs. 41%, p = 0.067).

Other studies have examined the activity of the related HMA decitabine in CMML. One of the first reports was published by the MDACC group on 19 patients with CMML, and a complete response rate of 58% was found. The dose of decitabine was 100 mg/m<sup>2</sup> per course, given in three different treatment schedules [57]. In a phase 2 trial from the GFM, 39 patients with advanced CMML were treated with decitabine at a dose of 20 mg/m<sup>2</sup> for 5 days. The median number of treatment cycles was 10 and the overall response rate 38%, OS at 2 years was 48%. Interestingly, the presence of *ASXL1* mutations had no significant impact on response or survival in that study [58]. A review of CMML patients that were included in several phase 2 and one phase 3 trials of decitabine in MDS was done by Wijermans et al. [59]. Among a total of 271 patients, 31 CMML patients were identified. The overall response rate was 25% with 14% CR, and 39% had stable disease. The treatment schedule was different than in the GFM study, and many patients received only a few cycles of therapy (median of four cycles).

Reliable and easily available predictors of response to treatment with HMA have not yet been identified. Although a correlation of response with *TET2* mutational status would seem plausible, this was not found in two different studies [58, 60]. Likewise, no other somatic mutation frequent in CMML proved to be predictive of response.

A small retrospective analysis has shown activity of decatibine and azacitidine in reducing spleen size in CMML patients. Spleen size was measured by physical examination, and complete or partial spleen response was found in 5 of 11 patients (45%) [61].

Although these data are promising and have led to the registration of both drugs for the treatment of CMML, data from phase 3 trials demonstrating a survival benefit are not yet available. Also, the optimal treatment schedule and treatment duration need to be defined. The results of an ongoing randomised trial (DACOTA trial) comparing hydroxyurea to decitabine in patients with advanced proliferative CMML are, therefore, eagerly awaited. This trial is conducted in three countries (France, Italy and Germany) and will include about 160 patients. The primary endpoint is progression‐free survival.

#### **7.5. Investigational agents**

response rate (60 vs. 36%) and OS (20 vs. 9 months) and has thus remained the treatment of choice for palliative cytoreduction in CMML patients [45]. The experience with intensive chemotherapy in CMML has been disappointing [53]. AML‐like induction therapy is only rarely used, usually as a preparatory regimen before allogeneic stem cell transplantation in

The introduction of the HMAs azacitidine and decitabine is likely to transform clinical management of CMML. A growing number of studies have shown considerable single agent activity with very low toxicity. In the largest study so far (76 patients from France, Cleveland Clinic and Lee Moffitt Cancer Center), a response rate of 43%, with 17% complete remissions, was found [54]. Of note, 46% had a proliferative form of CMML, as defined by a leukocyte

palpable splenomegaly had a negative impact on survival. A smaller Italian retrospective study analysed the response in 31 patients with CMML (42% with CMML‐1, 58% with CMML‐

rate was 51%, including 45% achieving complete remission [55]. A study from the Austrian Azacitidine registry reported on the outcome of 48 patients treated with azacitidine at 11 different centres [56]. Mean age was 71 years; 40% had CMML‐1; 60% had CMML‐2; and splenomegaly was found in 48%. Even in this unselected cohort with several high‐risk features, there was a surprising response rate of 70%, including 22% complete responses. Matched paired analysis suggested a better 2‐year‐survival when compared to best supportive care (62

Other studies have examined the activity of the related HMA decitabine in CMML. One of the first reports was published by the MDACC group on 19 patients with CMML, and a complete response rate of 58% was found. The dose of decitabine was 100 mg/m<sup>2</sup> per course, given in three different treatment schedules [57]. In a phase 2 trial from the GFM, 39 patients with

number of treatment cycles was 10 and the overall response rate 38%, OS at 2 years was 48%. Interestingly, the presence of *ASXL1* mutations had no significant impact on response or survival in that study [58]. A review of CMML patients that were included in several phase 2 and one phase 3 trials of decitabine in MDS was done by Wijermans et al. [59]. Among a total of 271 patients, 31 CMML patients were identified. The overall response rate was 25% with 14% CR, and 39% had stable disease. The treatment schedule was different than in the GFM study, and many patients received only a few cycles of therapy (median of four cycles).

Reliable and easily available predictors of response to treatment with HMA have not yet been identified. Although a correlation of response with *TET2* mutational status would seem plausible, this was not found in two different studies [58, 60]. Likewise, no other somatic

A small retrospective analysis has shown activity of decatibine and azacitidine in reducing spleen size in CMML patients. Spleen size was measured by physical examination, and

2) who were treated with azacitidine at a dose of 75 or 50 mg/m<sup>2</sup>

advanced CMML were treated with decitabine at a dose of 20 mg/m<sup>2</sup>

mutation frequent in CMML proved to be predictive of response.

complete or partial spleen response was found in 5 of 11 patients (45%) [61].

/L. In that study, the presence of more than 10% bone marrow blasts and

for 7 days. The overall response

for 5 days. The median

patients with an aggressive disease.

**7.4. Hypomethylating agents**

count of >13 × 10<sup>9</sup>

172 Myelodysplastic Syndromes

vs. 41%, p = 0.067).

A large number of investigational agents have been tested in CMML, among them tyrosine kinase inhibitors, farnesyltransferase inhibitors, immunomodulators and most recently JAK2 inhibitors. The experience with many of these approaches is limited to small phase 1 or phase 2 studies that have not been further developed, either because of limited activity or because of significant toxicity.

Imatinib has shown no effect in CMML patients without a *PDGFRB* rearrangement. Because CMML cells often have *RAS* activating mutations, drugs targeting this pathway have been tested. In a phase 2 trial, 35 patients with CMML were treated with lonafarnib (200–300 mg twice daily), one CR and 7 haematological improvements were reported. Major toxicities were gastrointestinal, fatigue, fever and hypokalemia [62]. Similar results were observed for tipifanib in a study of 10 CMML patients [63]. In several patients treated with lonafarnib, a significant increase in the white blood cell count was noted, sometimes accompanied by oedema and respiratory symptoms. This complication resolved quickly after discontinuation of lonafarnib and treatment with dexamethasone [64]. Disappointingly, translational studies have shown no correlation between responses and inhibition of farnesyl transferase.

Interesting results have been found in a study targeting angiogenesis in MDS and CMML patients with a combination consisting of melphalan (2 mg/day) and lenalidomide (10 mg/ day). Changes in circulating endothelial cells and plasma VEGF levels served as biomarkers of angiogenesis. The response rate was 33% in CMML patients (3/9), all of which had a proliferative form of the disease. Interestingly, there was a correlation between response and angiogenesis inhibition in these patients. Dose reductions were frequently necessary, but many patients were cytopenic already at baseline [65].

Most recently, a multicentre phase 1 trial (only published in abstract form) tested the JAK2 inhibitor ruxolitinib in 19 CMML patients. All patients had CMML‐1 and those with significant cytopenias were excluded. No dose limiting toxicity was noted. Although there were few haematologic responses, a frequent improvement of splenomegaly and B symptoms was found. A phase 2 trial testing ruxolitinib at a dose of 20 mg BID is planned [66].

#### **8. Summary and outlook**

CMML is a rare myeloid neoplasm with an overall poor prognosis. Important progress has been made in recent years in several aspects. First, the recognition of CMML as a unique disease entity, separated from the myelodysplastic syndromes, is an important step towards optimis‐ ing clinical management. Second, the introduction of CPSSs will improve patient selection in clinical trials. Phase 3 clinical trials in CMML patients will soon define the role of HMA in treatment. The elucidation of the mutational landscape in CMML has not provided disease‐ specific mutations but highly characteristic mutational combinations, particularly of *TET2* and *SRSF2*. These insights into molecular pathology are very likely to provide the basis for the development of novel therapeutic agents. Individualised therapies based on the predominant gene mutations could be envisaged. For example, while patients with *TET2* mutations are treated with HMA, in patients with mutations affecting signalling, specific pathway inhibitors might be more potent. Clearly, novel strategies and agents are needed for this still difficult to treat disease.

#### **Author details**

Andreas Himmelmann\*

Address all correspondence to: andreas.himmelmann@hirslanden.ch

Haematology Practice Lucerne, Clinic St. Anna, Lucerne, Switzerland

#### **References**


[7] Voglová J, Chrobák L, Neuwirtová R, Malasková V, Straka L. Myelodysplastic and myeloproliferative type of chronic myelomonocytic leukemia—distinct subgroups or two stages of the same disease? Leuk Res 2001;25:493–9.

entity, separated from the myelodysplastic syndromes, is an important step towards optimis‐ ing clinical management. Second, the introduction of CPSSs will improve patient selection in clinical trials. Phase 3 clinical trials in CMML patients will soon define the role of HMA in treatment. The elucidation of the mutational landscape in CMML has not provided disease‐ specific mutations but highly characteristic mutational combinations, particularly of *TET2* and *SRSF2*. These insights into molecular pathology are very likely to provide the basis for the development of novel therapeutic agents. Individualised therapies based on the predominant gene mutations could be envisaged. For example, while patients with *TET2* mutations are treated with HMA, in patients with mutations affecting signalling, specific pathway inhibitors might be more potent. Clearly, novel strategies and agents are needed for this still difficult to

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## **Different Mechanisms of Drug Resistance in Myelodysplastic Syndromes and Acute Myeloid Leukemia**

Lucia Messingerova, Denisa Imrichova, Martina Coculova, Marian Zelina, Lucia Pavlikova, Helena Kavcova, Mario Seres, Viera Bohacova, Boris Lakatos, Zdena Sulova and Albert Breier

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63483

#### **Abstract**

Myelodysplastic syndromes (MDSs) represent clonal hematopoietic stem cell (HSC) disorders in which genetic and/or epigenetic alteration are involved in the normal function of hematopoietic stem and progenitor cells. This results in the development of blood cytopenias and bone marrow dysplasia. In recent years, therapy with hypomethylating agents (HMAs) in combination with supportive therapies is recommended as frontline treatment for patients with high-risk MDSs according to International Prognostic Scoring System (IPSS HR-MDS). Therapy with HMAs is essential namely for IPSS HR-MDS patients who do not proceed to immediate allogeneic stem cell transplantation (al‐ loSCT). For IPSS LR-MDS (International Prognostic Scoring System, low-risk MDSs) patients, however, supportive therapies and growth factors are the mainstay of treatment. Some patients in this group are treated with immunomodulatory agents derived from thalidomide (lenalidomide) or using immunosuppressive therapy (IST). The therapeu‐ tic decisions can change during the course of the disease based on changes in riskcategory and the functional status of patients, in response to prior therapies, changes in patient preferences, and other factors.

Resistance to chemotherapy is a serious obstacle to the successful treatment of overall malignancies, including AML and MDS. The failure of therapeutic treatment may be due to the development of multidrug resistance (MDR) phenotype. MDR represents the induction of large-scale defensive mechanisms from which the upregulation of membrane transporters (like P-glycoprotein – P-gp) effluxing chemotherapeutic drugs from tumor cells represents the most observed molecular causality. Other mechanisms of MDR include drug metabolism, alterations in drug-induced apoptosis, epigenetic changes, epithelial-

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

mesenchymal transition, alteration in drug targets structures, and acceleration of DNA repair.

The present contribution represents a state-of-the-art review of available knowledge about this issue.

**Keywords:** myelodysplastic syndromes, acute myeloid leukemia, multidrug resist‐ ance, lenalidomide, 5-azacytidine, 5-aza-2-deoxyazacytidine

#### **1. Introduction**

Myelodysplastic syndromes (MDSs) represent the group of disorders associated with altered hematopoietic stem cells (HSCs) that lead to inefficient hematopoiesis [1]. This clinically results in dysplasia in one or more myeloid cell lineages, and variable degrees of cytopenias. The mean age of MDS patients' diagnosis ranges from 60 to 70 years. The incidence of MDS varied from 4.3 to 1.8 per 100,000 individuals per year in the US and Europe, respectively. Incidence slightly favors Caucasian males. MDS can lead to acute myeloid leukemia (AML) in 10–15% of patients.

Improvements in cytogenetic analysis techniques enable predicting the risk of MDS patients lapse into AML and the selection of optimal therapy [2]. The International Prognostic Scoring System (IPSS) described in the 1990s [3] is still commonly used. This scoring system defines how to measure the risk of patients' development from MDS to AML, and recommends dividing patients into four groups (low, intermediate 1, intermediate 2, and high risk). In lower risk patients, a combination of supportive care (includes transfusions of blood products, antibiotics) with substance improving erythropoiesis, immunosuppressive therapy, immuno‐ modulatory therapy, and stem cell transplantation has been used. Treatment options for patients diagnosed as higher risk include demethylating agents, cytotoxic chemotherapy, bone marrow HSC transplantation, and experimental treatments in clinical trials [4].

#### **2. Treatment options**

The only curative option for patients with MDS represents hematopoietic bone marrow stem cell transplantations. However, alloSCT is not available for all patients because of the comor‐ bidities of elderly patients [1]. Therefore some patients cannot be treated with alloSCT and other treatment options have to be used.

#### **2.1. IPSS low-risk MDS patients' treatment option**

Supportive care is an important therapy for the management of patients with low-risk MDS, as well as patients with poor disease prognosis which due to age or physical condition could not be treated with more intensive forms of therapy [5]. Several low-risk patients are dependent on blood transfusions. However, patients treated with blood transfusions may be overloaded with iron ions, so that iron chelation therapy is required [6]. Due to the partial dysfunction of immunity, antibiotics are needed for treatable infections [7].

There are three commonly used therapies for low-risk MDS patients: i. erythropoiesisstimulating agents (ESAs); ii. immunosuppressive therapy; and iii. immunomodulatory therapy with thalidomide derivative lenalidomide (revlimid). The treatment of patients with ESAs leads to significant erythroid response in 20–70% of unselected patients with MDS [1]. A median response for treatment with erythropoietin and colony-stimulating factor (CSF) applied together was 2 years and improves life quality [8]. Several immunosuppressants such as antithymocyte globulin and cyclosporine A were studied in a randomized phase III clinical trial. This treatment seems to be associated with hematologic responses in a subset of patients, however, it was not found to reduce the 2-year transformation and overall survival [9].

Over recent years, attention has been paid to immunomodulatory-acting drugs (IMIDs). The anti-MDS activity of these drugs involves antiproliferative effects, downregulation of crucial cytokines, and costimulatory effects on T and NK cells [10]. The IMIDs are thalidomide analogs which have greater immunological and anticancer properties, but lack the toxicity associated with thalidomide [11]. Lenalidomide (LEN) was proven to be effective in the treatment of patients with low-risk MDS, particularly in cases with special molecular feature, i.e., deletions in the long arm of chromosome 5 [12, 13].

#### **2.2. IPSS high-risk MDS patients' treatment option**

mesenchymal transition, alteration in drug targets structures, and acceleration of DNA

The present contribution represents a state-of-the-art review of available knowledge

**Keywords:** myelodysplastic syndromes, acute myeloid leukemia, multidrug resist‐

Myelodysplastic syndromes (MDSs) represent the group of disorders associated with altered hematopoietic stem cells (HSCs) that lead to inefficient hematopoiesis [1]. This clinically results in dysplasia in one or more myeloid cell lineages, and variable degrees of cytopenias. The mean age of MDS patients' diagnosis ranges from 60 to 70 years. The incidence of MDS varied from 4.3 to 1.8 per 100,000 individuals per year in the US and Europe, respectively. Incidence slightly favors Caucasian males. MDS can lead to acute myeloid leukemia (AML) in 10–15% of patients.

Improvements in cytogenetic analysis techniques enable predicting the risk of MDS patients lapse into AML and the selection of optimal therapy [2]. The International Prognostic Scoring System (IPSS) described in the 1990s [3] is still commonly used. This scoring system defines how to measure the risk of patients' development from MDS to AML, and recommends dividing patients into four groups (low, intermediate 1, intermediate 2, and high risk). In lower risk patients, a combination of supportive care (includes transfusions of blood products, antibiotics) with substance improving erythropoiesis, immunosuppressive therapy, immuno‐ modulatory therapy, and stem cell transplantation has been used. Treatment options for patients diagnosed as higher risk include demethylating agents, cytotoxic chemotherapy, bone

The only curative option for patients with MDS represents hematopoietic bone marrow stem cell transplantations. However, alloSCT is not available for all patients because of the comor‐ bidities of elderly patients [1]. Therefore some patients cannot be treated with alloSCT and

Supportive care is an important therapy for the management of patients with low-risk MDS, as well as patients with poor disease prognosis which due to age or physical condition could

marrow HSC transplantation, and experimental treatments in clinical trials [4].

ance, lenalidomide, 5-azacytidine, 5-aza-2-deoxyazacytidine

repair.

182 Myelodysplastic Syndromes

**1. Introduction**

**2. Treatment options**

other treatment options have to be used.

**2.1. IPSS low-risk MDS patients' treatment option**

about this issue.

Patients with high-risk MDS have poor outcomes, high probability of AML development, and without intensive treatment or alloSCT their median survival is limited to 1 year [3]. The treatment of high-risk MDS patients is based on three commonly used therapies: i. alloSCT; ii. intensive chemotherapy; and iii. drugs with epigenetic mechanism of action such as deme‐ thylation agents and histone deacetylase inhibitors (HDACi) [1]. Similarly as in low-risk patients, the application of alloSCT is limited by the patients' age and overall condition.

About 50% of patients with high-risk MDS achieve complete remission with standard anti‐ leukemic chemotherapy with fludarabine, idarubicin, or topotecan. This therapy could be improved by a combination of these drugs with intermediate- or high-dose cytosine arabino‐ side [14]. The combination of such therapy with granulocyte colony-stimulating factor (G-CSF) is well tolerated and highly effective in the remission of both high-risk MDS patients and AML patients [15].

Inhibitors of histone deacetylase block the deacetylation of histones molecules, i.e., they protect histones in acetylation forms. The acetylation of histones occurs in the replication- and transcription-active euchromatin. HDACi could protect euchromatin against formation changes to heterochromatin that is replication and transcription inactive. The effect of HDACi could be pleiotropic, leading to the induction of differentiation, growth arrest, and finally to the apoptosis of tumor cells. The mechanisms of HDACi's effectiveness are under intensive debate, and it may be p53 dependent or independent [16]. Valproic acid, entinostat, vorinostat, and other HDCAi are under intensive research with the aim of characterizing their effective‐ ness against MDS [16].

The cytosine analogs 4-amino-1-(β-D-ribofuranosyl)-1,3,5-triazin-2(1H)-one – azacytidine (AzaC) and 4-amino-1-(β-D-deoxyribofuranosyl)-1,3,5-triazin-2(1H)-one – deoxyazacytidine (decitabine, DAC), which were described as cancerostatic agents in the late 1960s and the early 1970s [17, 18], were found to effectively block DNA methylation [19]. Their effectiveness in inducing beneficial effects in the treatment of MDS [20] and AML [21] was already proven. The downregulation of DNA methylation induced by AzaC and DAC is related to the ability of this substance to be artificially incorporated into DNA instead of cytosine, which has to be methylated by DNA methyltransferase [22]. This could be considered as a major principle of DAC action. In contrast to DAC's effects, AzaC is more complex and also involves incorpora‐ tion into mRNA, tRNA, and rRNA, which disrupts nucleic acid and protein metabolism leading to apoptosis in addition to the incorporation of substances into DNA [22, 23]. Consis‐ tently AzaC induced more pronounced cell damage effects than DAC [24].

#### **3. Drug resistance of MDS and AML patients**

#### **3.1. Mechanisms of drug resistance of neoplastic cells**

The multidrug resistance (MDR) of neoplastic cells represents a real obstacle in the effective treatment of neoplastic diseases [25]. MDR could be an inherent property of tissue from which neoplastic cells were developed – primary (intrinsic) MDR, or could be induced by prior treatment with anticancer drugs – secondary (acquired) MDR (reviewed in [26]). In both cases the neoplastic cells exert reduced sensitivity to more than one drug that differs in structure and pharmacological efficiency. In many cases, cells with resistance to a large scale of diverse drugs are present in cancer tissue. Several mechanisms are involved in the mediation of MDR, which can be divided into seven groups (**Figure 1**, reviewed in [27]):


Different Mechanisms of Drug Resistance in Myelodysplastic Syndromes and Acute Myeloid Leukemia http://dx.doi.org/10.5772/63483 185

debate, and it may be p53 dependent or independent [16]. Valproic acid, entinostat, vorinostat, and other HDCAi are under intensive research with the aim of characterizing their effective‐

The cytosine analogs 4-amino-1-(β-D-ribofuranosyl)-1,3,5-triazin-2(1H)-one – azacytidine (AzaC) and 4-amino-1-(β-D-deoxyribofuranosyl)-1,3,5-triazin-2(1H)-one – deoxyazacytidine (decitabine, DAC), which were described as cancerostatic agents in the late 1960s and the early 1970s [17, 18], were found to effectively block DNA methylation [19]. Their effectiveness in inducing beneficial effects in the treatment of MDS [20] and AML [21] was already proven. The downregulation of DNA methylation induced by AzaC and DAC is related to the ability of this substance to be artificially incorporated into DNA instead of cytosine, which has to be methylated by DNA methyltransferase [22]. This could be considered as a major principle of DAC action. In contrast to DAC's effects, AzaC is more complex and also involves incorpora‐ tion into mRNA, tRNA, and rRNA, which disrupts nucleic acid and protein metabolism leading to apoptosis in addition to the incorporation of substances into DNA [22, 23]. Consis‐

The multidrug resistance (MDR) of neoplastic cells represents a real obstacle in the effective treatment of neoplastic diseases [25]. MDR could be an inherent property of tissue from which neoplastic cells were developed – primary (intrinsic) MDR, or could be induced by prior treatment with anticancer drugs – secondary (acquired) MDR (reviewed in [26]). In both cases the neoplastic cells exert reduced sensitivity to more than one drug that differs in structure and pharmacological efficiency. In many cases, cells with resistance to a large scale of diverse drugs are present in cancer tissue. Several mechanisms are involved in the mediation of MDR,

**i.** Potentiating drug metabolism via the induction/activation of phase I and phase II

**ii.** Potentiating cell drug efflux via the induction/activation of membrane drug trans‐

tently AzaC induced more pronounced cell damage effects than DAC [24].

which can be divided into seven groups (**Figure 1**, reviewed in [27]):

porter predominantly members of the ABC family

**3. Drug resistance of MDS and AML patients**

**3.1. Mechanisms of drug resistance of neoplastic cells**

detoxification enzymes

**iii.** Alteration in drug target structures

**v.** Changes in epigenetic regulation

**vi.** Programmed cell death inhibition

**vii.** Epithelial-mesenchymal transition

**iv.** Acceleration of DNA-repair

ness against MDS [16].

184 Myelodysplastic Syndromes

These mechanisms could act independently or cooperate in the development of MDR in relation to cancer cells' specific character. The expression of drug transporters represents the most observed molecular causality of MDR (reviewed in [28, 29]). At least three transporters are involved in the reduction of drug sensitivity of neoplastic cells. The best known is Pglycoprotein (P-gp) that represents an ABCB1 member of the ABC transporter family and was discovered as the first ABC transporter in 1976 [30]. P-gp could efflux a large scale of different uncharged substances from cells. Drugs such as colchicine, tacrolimus, and quinidine; chemotherapeutic agents such as etoposide, doxorubicin, and vinblastine; different lipids and steroids; xenobiotics; DNA-intercalators such as ethidium bromide; linear or circular peptides like valinomycin and gramicidin; bilirubin; cardiac glycosides like digoxin; different immu‐ nosuppressive agents; glucocorticoids like dexamethasone; HIV-type 1 antiretroviral therapy agents like protease inhibitors and nonnucleoside reverse transcriptase inhibitors; and many others are known to be P-gp substrates. When P-gp is expressed in neoplastic tissue it can depress cell sensitivity to its substrates several hundred times [31]. Besides this generally accepted role as a drug transporter, this protein may also play another role as an antiapoptotic regulatory protein and this role is independent of P-gp efflux activity [29, 32]. This additional role also enables P-gp to reduce cell sensitivity to substances that are not its substrates, such as cisplatin several times [33, 34].

Other important transporters involved in MDR are ABCC1-3 members of the ABC transport‐ ers' family, also known as multidrug-resistant associate proteins 1–3 (MRP1-MRP3) that in contrast to P-gp are specific to negatively charged organic anions (reviewed in [35]). They are also specific for drug conjugates with glucuronic acid and glutathione as a product of phase II enzyme drug detoxification.

One more transporter ABCG2 member of the ABC transporter family is often described to be involved in MDR [36]. This transporter also known as breast cancer-resistant protein (BCRP) may efflux substances such as mitoxantrone, methotrexate, topotecan, imatinib, and others. The substrate specificity of P-gp, MRP1-3, and BCRP overlaps, and each could be responsible for the efflux of common substrate.

The drug could be detoxified by phase I and phase II detoxification enzymes that secure oxidative and conjugative ways of drug modification which are mediated by cytochrome P450 (CYP) monooxygenases and conjugating enzymes (glutathione S-transferases [GSTs] and UDP-glucuronyl-transferase), respectively [37]. The CYP family, particularly the CYP3A members of the CYP family, may be involved in the reduction of cell sensitivity to several drugs. The transcriptional control of the CYP family is mediated by pregnane X nuclear receptor, i.e., the same nuclear receptor involved in P-gp expression [38].

GSTs represent a group of enzymes that are often involved in the protection of cells against toxic stress [39]. These enzymes catalyze the conjugation of several xenobiotics with reduced glutathione [40]. The actions of GSTs are often coordinated with MRPs that transport several conjugates of drugs and reduced glutathione [41]. While P-gp cannot transport glutathione conjugates, coordinated coexpression of P-gp and GST was observed in vitro using AML cell lines [42].

Alteration in drug target structures such as the mechanism of MDR represents a large group of diverse changes in regulatory pathways, which is finally responsible for the downregulation or upregulation of drug molecular target. An example of this behavior alteration of topoiso‐ merase II, such as the molecular causality of neoplastic cell resistance to topoisomerase poisons, could be performed (reviewed in [28]).

The repair of DNA primarily damaged by drugs' direct action, or secondarily by the elevation of oxygen reactive species formation, clearly yields to the drug resistance of neoplastic cells [27]. The therapeutic effects of DNA-damaging drugs in cancer treatment are given by the equilibrium between drug-induced DNA damage and the effectiveness of DNA repair mechanisms. The inhibition of repair pathways used in conjunction with DNA damaging chemotherapy could sensitize cancer cells and therefore increase the efficacy of therapy [27].

Epithelial-mesenchymal transition is a mechanism predominantly taking part in solid tumor metastatic processes. This mechanism could play only a minor role (if any) in drug resistance development in MDS and AML patients.

The high expression levels of antiapoptotic proto-oncogene of the Bcl-2 family (such as BCL-2, Bcl-XL, Mcl-1, Bcl-w, and Bfl-1) were often reported to be associated with in vitro resistance to chemotherapeutic agents, poor clinical outcomes in cases of AML [43], and in cases of adults with acute lymphoblastic leukemia [44]. Bcl-2 was shown to be restricted in tissues character‐ ized by apoptotic cell death [45]. Antiapoptotic proteins of the Bcl-2 family hetero-oligomerize in vivo with a conserved homolog – proapoptotic member of the Bcl-2 family (such as Bax), and this process is known to modulate apoptosis [46]. The translocation of the Bax (or other proapoptotic protein) monomer from the cytosol to the mitochondria followed by the forma‐ tion of BAX homo-oligomers represents a physiological death stimulus, which may be prevented by the presence of the Bcl-2 protein (or other antiapoptotic proteins) [47]. Therefore for apoptosis progression, an equilibrium between anti- and proapoptotic proteins plays a crucial role. This is molecularly regulated by the p53 known as the central regulator of apoptosis [48]. This is consistent with known data about the role of the mutated form of TP53 in cancer [49, 50].

Epigenetic regulations are involved in the development of MDR directly by the downregula‐ tion or upregulation of important genes responsible for cell death or survival. For example, tumor-suppressor genes are often silenced via hypermethylation, and oncogenes are overex‐ pressed via hypomethylation [27]. Epigenetics could also play an indirect role in cell drug sensitivity by the following mechanism: the opening of the chromatin structure, which is prerequisite for DNA replication and transcription, and to produce uncovered DNA that is more accessible for drug-induced DNA damage. This is consistent with more pronounced DNA damage induced with drugs in more proliferating and/or transcriptionally active cells.

Hypermethylation of the MDR1 promoter was associated with transcriptional repression and chromatin structural changes [51]. The demethylation of this promoter in cancer cell lines was found to elevate the multidrug-resistant phenotype [52].

Epigenetic mechanisms can also influence DNA damage repair. For example, DNA mismatch repair processes can be lost due to the hypermethylation of the human mutL homolog 1 (hMLH1) gene promoter, which can lead to cancer development [27].

Demethylation by DAC may have a role in increasing the efficacy of chemotherapy for patients with tumors, as characterized by high hMLH1 promoter methylation and low hMLH1 expression [53].

#### **3.2. Resistance to immunomodulatory drugs**

may efflux substances such as mitoxantrone, methotrexate, topotecan, imatinib, and others. The substrate specificity of P-gp, MRP1-3, and BCRP overlaps, and each could be responsible

The drug could be detoxified by phase I and phase II detoxification enzymes that secure oxidative and conjugative ways of drug modification which are mediated by cytochrome P450 (CYP) monooxygenases and conjugating enzymes (glutathione S-transferases [GSTs] and UDP-glucuronyl-transferase), respectively [37]. The CYP family, particularly the CYP3A members of the CYP family, may be involved in the reduction of cell sensitivity to several drugs. The transcriptional control of the CYP family is mediated by pregnane X nuclear

GSTs represent a group of enzymes that are often involved in the protection of cells against toxic stress [39]. These enzymes catalyze the conjugation of several xenobiotics with reduced glutathione [40]. The actions of GSTs are often coordinated with MRPs that transport several conjugates of drugs and reduced glutathione [41]. While P-gp cannot transport glutathione conjugates, coordinated coexpression of P-gp and GST was observed in vitro using AML cell

Alteration in drug target structures such as the mechanism of MDR represents a large group of diverse changes in regulatory pathways, which is finally responsible for the downregulation or upregulation of drug molecular target. An example of this behavior alteration of topoiso‐ merase II, such as the molecular causality of neoplastic cell resistance to topoisomerase

The repair of DNA primarily damaged by drugs' direct action, or secondarily by the elevation of oxygen reactive species formation, clearly yields to the drug resistance of neoplastic cells [27]. The therapeutic effects of DNA-damaging drugs in cancer treatment are given by the equilibrium between drug-induced DNA damage and the effectiveness of DNA repair mechanisms. The inhibition of repair pathways used in conjunction with DNA damaging chemotherapy could sensitize cancer cells and therefore increase the efficacy of therapy [27]. Epithelial-mesenchymal transition is a mechanism predominantly taking part in solid tumor metastatic processes. This mechanism could play only a minor role (if any) in drug resistance

The high expression levels of antiapoptotic proto-oncogene of the Bcl-2 family (such as BCL-2, Bcl-XL, Mcl-1, Bcl-w, and Bfl-1) were often reported to be associated with in vitro resistance to chemotherapeutic agents, poor clinical outcomes in cases of AML [43], and in cases of adults with acute lymphoblastic leukemia [44]. Bcl-2 was shown to be restricted in tissues character‐ ized by apoptotic cell death [45]. Antiapoptotic proteins of the Bcl-2 family hetero-oligomerize in vivo with a conserved homolog – proapoptotic member of the Bcl-2 family (such as Bax), and this process is known to modulate apoptosis [46]. The translocation of the Bax (or other proapoptotic protein) monomer from the cytosol to the mitochondria followed by the forma‐ tion of BAX homo-oligomers represents a physiological death stimulus, which may be prevented by the presence of the Bcl-2 protein (or other antiapoptotic proteins) [47]. Therefore for apoptosis progression, an equilibrium between anti- and proapoptotic proteins plays a

receptor, i.e., the same nuclear receptor involved in P-gp expression [38].

for the efflux of common substrate.

186 Myelodysplastic Syndromes

poisons, could be performed (reviewed in [28]).

development in MDS and AML patients.

lines [42].

Over recent years, attention has been paid to exploiting the immunomodulatory effects primarily obtained for thalidomide [54], which has resulted in novel IMIDs. The anti-MDS activity of these drugs (namely LEN) was proven for low-risk MDS, particularly with 5q deletion (del[5q]) [55]. This action is attributed to several mechanisms that involve antiproli‐ ferative effects, downregulation of crucial cytokines, and costimulatory effects on T and NK cells [10]. However, the exact mechanism of IMIDs' action in MDS treatment is still not fully understood. The IMIDs' immunomodulatory compounds derived from the thalidomide structure have greater immunological and anticancer properties, but lack the toxicity associ‐ ated with thalidomide [11]. LEN (Revlimid) was approved for use in low- and intermediate-1 risk MDS patients who have the deletion 5q chromosome and no other chromosomal abnormalities, are dependent on red blood cell transfusions, and for whom other treatment options have been found to be insufficient or inadequate by EMA (European Medicines Agency) and the FDA (Food and Drug Administration). After LEN treatment, blood transfu‐ sion-independent rates were 56–67%, and median response duration was longer than 104 weeks [1]. Additionally, a significant proportion of these responders achieved cytogenetic responses (50–76%), indicating a direct cytotoxic effect of LEN on the neoplastic clones, although a significant proportion of patients develop resistance to this treatment. The study of cytogenetics and molecular predictors of responses in patients with myeloid malignancies without del[5q] treated with LEN indicated that treatment could be effective in patients with

normal karyotype and a gain of 8 chromosome is present [55]. The LEN response was achieved by one quarter of MDS patients lacking the 5q abnormality. Ebert et al. [56] found that mononuclear cells from bone marrow aspirates of patients who respond to LEN have a decreased expression of genes, which are specific to terminal erythroid differentiation, regardless of the presence or absence of a 5q deletion. Moreover, LEN acts directly on hema‐ topoietic progenitor cells to increase erythropoiesis relative to other lineages.

The mechanism of LEN's therapeutic effects and the mechanisms that depress its effectiveness in MDS treatment are not fully understood, but could be related to TP53 mutation (reviewed in [57]).

TP53-mutated populations seem to be associated with the early stage of low-risk MDS in patients with del[5q] [58]. However, these authors stated that TP53 mutations could not be predicted by general clinical features but were associated with p53 overexpression. Specific R72P polymorphism of TP53 results in two molecular forms of p53. Molecular variant p53-R72 with better mitochondrial localization activates apoptosis more efficiently (by direct induction of cytochrome c release) than p53-P72 variant [59]. McGraw et al. [60] underscore the distri‐ bution of R72P in MDS and highlight differences between del(5q) and non-del(5q) subtypes by gene polymorphism and the relationship to LEN response. However, to prove the potential interaction of R72P variants with germline variants in other key regulators or effectors of the p53 pathway that may modify MDS risk and LEN treatment response, further research will be necessary.

Allelic deletion of the RPS14 gene is a key effector of the hypoplastic anemia in patients with MDS and chromosome 5q deletion [61]. Disruption of ribosome integrity liberates free ribosomal proteins to bind to and trigger the degradation of E3 ubiquitin-protein ligase MDM2 (a negative regulator of p53), with consequent p53 transactivation. Consistently, p53 is overexpressed in erythroid precursors of primary bone marrow del(5q) MDS specimens accompanied by reduced cellular MDM2. LEN may act in the stabilization of MDM2 that leads to p53 degradation [61].

When LEN was used in establishing human AML cell lines SKM-1 and MOLM-13 for resist‐ ance, only SKM-1 but not MOLM-13 cells developed MDR phenotype with massive expression of P-gp [62]. Both these cell lines were derived from AML patients, whose disease developed from MDS. In contrast to MOLM-13 with wild type of p53 [63, 64], SKM-1 represents cells expressing a mutated p53 form [65]. Thus cells with mutated TP53 could express P-gp under long-term LEN treatment, which leads to typical P-gp mediated MDR.

#### **3.3. Resistance to hypomethylating agents**

In pathogenesis of MDS, both genetics and epigenetics alterations are cooperated. Disruption of genetic pathways regulating the processes of self-renewal, differentiation, quiescence, and stem cell-niche signaling contributes to AML transformation. The hypermethylation of different genes was discussed to be partially responsible for the poor prognosis of MDS patients [66]. Demethylating agents, such as AzaC and DAC, were shown to induce clinical responses in 40–70% of MDS patients [67, 68]. Although hypomethylation is considered the dominant mode of therapeutic action of these drugs, it may also induce DNA damage and consequent apoptosis [69]. Interestingly, clinically significant responses to decitabine without significant toxicity can be seen in patients after nonsuccessful azacitidine therapy [70].

normal karyotype and a gain of 8 chromosome is present [55]. The LEN response was achieved by one quarter of MDS patients lacking the 5q abnormality. Ebert et al. [56] found that mononuclear cells from bone marrow aspirates of patients who respond to LEN have a decreased expression of genes, which are specific to terminal erythroid differentiation, regardless of the presence or absence of a 5q deletion. Moreover, LEN acts directly on hema‐

The mechanism of LEN's therapeutic effects and the mechanisms that depress its effectiveness in MDS treatment are not fully understood, but could be related to TP53 mutation (reviewed

TP53-mutated populations seem to be associated with the early stage of low-risk MDS in patients with del[5q] [58]. However, these authors stated that TP53 mutations could not be predicted by general clinical features but were associated with p53 overexpression. Specific R72P polymorphism of TP53 results in two molecular forms of p53. Molecular variant p53-R72 with better mitochondrial localization activates apoptosis more efficiently (by direct induction of cytochrome c release) than p53-P72 variant [59]. McGraw et al. [60] underscore the distri‐ bution of R72P in MDS and highlight differences between del(5q) and non-del(5q) subtypes by gene polymorphism and the relationship to LEN response. However, to prove the potential interaction of R72P variants with germline variants in other key regulators or effectors of the p53 pathway that may modify MDS risk and LEN treatment response, further research will be

Allelic deletion of the RPS14 gene is a key effector of the hypoplastic anemia in patients with MDS and chromosome 5q deletion [61]. Disruption of ribosome integrity liberates free ribosomal proteins to bind to and trigger the degradation of E3 ubiquitin-protein ligase MDM2 (a negative regulator of p53), with consequent p53 transactivation. Consistently, p53 is overexpressed in erythroid precursors of primary bone marrow del(5q) MDS specimens accompanied by reduced cellular MDM2. LEN may act in the stabilization of MDM2 that leads

When LEN was used in establishing human AML cell lines SKM-1 and MOLM-13 for resist‐ ance, only SKM-1 but not MOLM-13 cells developed MDR phenotype with massive expression of P-gp [62]. Both these cell lines were derived from AML patients, whose disease developed from MDS. In contrast to MOLM-13 with wild type of p53 [63, 64], SKM-1 represents cells expressing a mutated p53 form [65]. Thus cells with mutated TP53 could express P-gp under

In pathogenesis of MDS, both genetics and epigenetics alterations are cooperated. Disruption of genetic pathways regulating the processes of self-renewal, differentiation, quiescence, and stem cell-niche signaling contributes to AML transformation. The hypermethylation of different genes was discussed to be partially responsible for the poor prognosis of MDS patients [66]. Demethylating agents, such as AzaC and DAC, were shown to induce clinical responses in 40–70% of MDS patients [67, 68]. Although hypomethylation is considered the

long-term LEN treatment, which leads to typical P-gp mediated MDR.

**3.3. Resistance to hypomethylating agents**

topoietic progenitor cells to increase erythropoiesis relative to other lineages.

in [57]).

188 Myelodysplastic Syndromes

necessary.

to p53 degradation [61].

Changes in the expression profile of genes like CD9, GPNMB, FUCA1, ANGPT1, PLA2G7, TPM1, and ARHGEF3 were observed when CD34+ cells isolated from the bone marrow of high-risk MDS patients treated in vitro with DAC were compared with CD34+ cells isolated from patients with untreated early stage Hodgkin's lymphoma taken as a control [66].

AzaC resistance represents a real obstacle for the effective treatment of MDS patients, which focused the attention of scientists on alternative therapeutic strategies for nonresponsive patients. For this reason, AzaC-resistant MDS/AML cell lines are established. AzaC-resistant SKM-1 cells exhibited increased expression of the BCL2L10 member of the antiapoptotic Bcl-2 family that altered apoptosis progression [71]. Interestingly we described the downregulation and changed molecular form of the Bcl-2 protein identified by polyclonal antibody (sc-492, Santa Cruz Biotechnology, USA) in our variant of SKM-1 AzaC-resistant cells [42]. Moreover, other AzaC-resistant AML cells derived from the MOLM-13 cell line exerted similar changes in the Bcl-2 protein. Significant correlation of AzaC resistance with a percentage of MDS or AML cells expressing BCL2L10 was established on a group of 77 patients [71].

AzaC resistance could include impaired mitochondrial membrane permeabilization and caspase activation when AzaC resistant and sensitive SKM-1 myeloid were compared [72]. In our experiments, when the same cell model was used resistance to AzaC was associated with strong over expression of P-gp that secured additional resistance to P-gp substrates [42]. This is a rather interesting finding because AzaC is not a P-gp substrate, and P-gp was not respon‐ sible for AzaC resistance. We obtained similar results using MOLM-13 cells. The activity of GST was found to be elevated 8 times when AzaC resistant and sensitive SKM-1 and MOLM-13 cells were compared [42].

AzaC induced the upregulation of LC3-II and elevation of cathepsin B activity (both autophagy markers). Increased basal autophagy was observed in SKM-1 AzaC-resistant cells, but these cells were resistant to AzaC-mediated autophagy [72]. Autophagy depression using a LC3 silencer revealed the protective function of autophagy in AzaC-sensitive and AzaC-resistant cells in basal condition [72]. Taking all the facts about apoptosis and autophagy progression in AML cell models together, it could be concluded that resistance to AzaC is associated with alterations of both processes via impaired homeostasis of its key regulators such as Bcl-2 family proteins and LC3. However, the exact mechanism of this feature is not fully understood and future research will be necessary.

Enzymes involved in cytidine metabolism such as cytidine deaminase (CDA) and deoxycyti‐ dine kinase (DCK) seem to be responsible for the primary (intrinsic) AzaC resistance, because nonresponders of AzaC have a 3-fold higher CDA/CDK ratio. There were no significant differences at relapse in DAC metabolism genes, and no CDK mutations were detected [73].

MDSs are characterized by mutations in genes encoding epigenetic modifiers and aberrant DNA methylation. Clonal mutation of TP53 and non-receptor type 11 protein tyrosine phosphatase were associated with shorter overall survival, but not the drug response of patients. Clonal tet methylcytosine dioxygenase 2 (TET2) mutations predicted a response when subclones were treated as wild type. The highest response rate was observed in patients with a mutation in the TET2 gene without a clonal mutation of the trancriptional regulator encoded by ASXL1 gene. [74].

While somatic mutations did not differentiate responders from nonresponders for DAC treatment, differentially methylated regions of DNA at baseline distinguished responders from nonresponders. In responders, the upregulated genes included those that are associated with the cell cycle, potentially contributing to effective DAC incorporation [75].

While DAC is generally accepted as a hypomethylating agent, it may exert a therapeutic effect also in another way. The acceleration of reactive oxygen species induced with DAC could take place in the overall DAC effect. However, reactive oxygen species accumulation was not always present in the sample of AML patients after DAC treatment. Therefore, the relevance of reactive oxygen species generation in the mechanism of DAC pharmacological effectiveness should be studied more intensively in the future [76].

#### **3.4. Resistance to intensive chemotherapy**

MDS intermediate- or high-risk patients may be treated with an intensive chemotherapy regimen that is similar as used for AML treatment. A combination of drugs such as cytosine arabinoside, fludarabine, idarubicin, and topotecan, etc. could be used [14]. This chemotherapy is oriented on destroying abnormal blood cells or preventing their growth. For patients who are eligible, bone marrow transplantation is recommended after this therapy.

The development of MDR resistance during the application of this protocol is similar as for other types of neoplastic diseases, and may involve each of the mechanisms described in Chapter 3. The combination of drug-resistant mechanisms that are included in MDR pheno‐ type development during high intensive treatment depends on the specific patient molecular feature, previous therapeutic history, and drugs applied during previous treatment.

#### **3.5. Resistance to CD33-targeted therapy**

Progressive methods of MDS treatment represent antibody targeting therapy with cytotoxic agents linked to humanized antibodies against antigens specific to neoplastic cells. Both MDS and AML are characterized by the presence of undifferentiated CD33-positive myeloblast in peripheral blood and bone marrow, compared with healthy control [77]. CD33 is a 67 kDa glycoprotein present on the surface of myeloid cells, and is a member of the sialic acid binding immunoglobulin-like lectin family of proteins [78]. After binding to an appropriate antibody, CD33 is rapidly internalized into leukemia cells [79, 80]. This action enables the use of a humanized CD33 antibody, conjugated to cytotoxic agents for targeted immunotherapy [78, 79]. Gemtuzumab ozogamicin represents antibody drug (from a class of calicheamicins) conjugates [7, 78–81]. This therapy has proven to be effective, but resistance to treatment could be developed. Another immune-targeting preparation is AVE9633 (immunoconjugate of humanized monoclonal CD33 antibody, linked through a disulfide bond to the maytansine derivative DM4) that was used in the treatment of several leukemia cell lines [82]. The activity of P-gp was attributed as a critical factor in depressing the success of this therapy. P-gp mediated the efflux of drug liberated from linkage with antibody due to intracellular enzymes was attributed as being responsible for this resistance [82]. However, a significant inverse correlation was determined for the expression of P-gp and CD33 in the AML blast obtained from patients [81]. We described the strong downregulation of CD33 on mRNA and the protein level in P-gp positive SKM-1 cells selected for resistance by LEN, vincristine, mitoxantrone, or in P-gp positive MOLM-13 cells selected for resistance by vincristine or mitoxantrone [62]. Upregulation of CD33 expression level was also observed in P-gp silenced cell lines. Therefore, the failure of CD33-targeted therapy of patients with AML blast overexpressing P-gp could be caused by a lack of CD33 as an antibody target structure on the cell surface.

#### **3.6. Detection of drug-resistant markers in neoplastic cells**

Cellular expression of drug-resistant markers could be monitored on mRNA level (RT-PCR methods) or protein level (Western blot and immunofluorescence flow cytometry) [83]. Proteins active in MDR development like ABC transporters, drug metabolizing enzymes, antiapoptotic proteins, and many others could be detected by these methods. Transport activity of drug transporters could be measured using depressed intracellular retention of their fluorescent substrates by flow cytometry. Retentions of calcein/AM, rhodamine 123, or doxorubicin were often used for P-gp efflux activity detection directly in cells isolated from patients [84]. Activities of detoxification enzymes could be monitored by appropriate sub‐ strates, which enzymatic modifications induce changes in either fluorescence or light absorb‐ ance properties. Conjugation of 1-chloro-2,4-dinitrobenzene with reduced glutathione as a result of GST activity could be taken as example [42]. Specific mutations of genes active in MDR development (like *TP53*) could be detected using mutation analysis including specifically designed PCR reaction, denaturing high-performance liquid chromatography, or DNAsequencing techniques. Alteration in drug-induced apoptosis could be monitored as difference in drug-induced DNA-fragmentation by electrophoresis or using comet assay [85]. Applica‐ tion of oligonucleotide microarray for human genome represents available methods to obtain complex information about expression profiles of MDR-associated genes in patients [86]. Moreover, when nonresponders and responders will be compared using oligonucleotide microarrays, new information about involving different genes' expression in MDR develop‐ ment could be obtained. Cytotoxic effects of several drugs could be monitored directly in cells isolated from patient samples using assays based on reduction of formazan (like MTT assay) by intracellular dehydrogenases, or liberation of fluorescent label from its esters (like fluores‐ cein diacetate) by intracellular esterases.

#### **4. Perspectives**

patients. Clonal tet methylcytosine dioxygenase 2 (TET2) mutations predicted a response when subclones were treated as wild type. The highest response rate was observed in patients with a mutation in the TET2 gene without a clonal mutation of the trancriptional regulator encoded

While somatic mutations did not differentiate responders from nonresponders for DAC treatment, differentially methylated regions of DNA at baseline distinguished responders from nonresponders. In responders, the upregulated genes included those that are associated

While DAC is generally accepted as a hypomethylating agent, it may exert a therapeutic effect also in another way. The acceleration of reactive oxygen species induced with DAC could take place in the overall DAC effect. However, reactive oxygen species accumulation was not always present in the sample of AML patients after DAC treatment. Therefore, the relevance of reactive oxygen species generation in the mechanism of DAC pharmacological effectiveness

MDS intermediate- or high-risk patients may be treated with an intensive chemotherapy regimen that is similar as used for AML treatment. A combination of drugs such as cytosine arabinoside, fludarabine, idarubicin, and topotecan, etc. could be used [14]. This chemotherapy is oriented on destroying abnormal blood cells or preventing their growth. For patients who

The development of MDR resistance during the application of this protocol is similar as for other types of neoplastic diseases, and may involve each of the mechanisms described in Chapter 3. The combination of drug-resistant mechanisms that are included in MDR pheno‐ type development during high intensive treatment depends on the specific patient molecular

Progressive methods of MDS treatment represent antibody targeting therapy with cytotoxic agents linked to humanized antibodies against antigens specific to neoplastic cells. Both MDS and AML are characterized by the presence of undifferentiated CD33-positive myeloblast in peripheral blood and bone marrow, compared with healthy control [77]. CD33 is a 67 kDa glycoprotein present on the surface of myeloid cells, and is a member of the sialic acid binding immunoglobulin-like lectin family of proteins [78]. After binding to an appropriate antibody, CD33 is rapidly internalized into leukemia cells [79, 80]. This action enables the use of a humanized CD33 antibody, conjugated to cytotoxic agents for targeted immunotherapy [78, 79]. Gemtuzumab ozogamicin represents antibody drug (from a class of calicheamicins) conjugates [7, 78–81]. This therapy has proven to be effective, but resistance to treatment could be developed. Another immune-targeting preparation is AVE9633 (immunoconjugate of humanized monoclonal CD33 antibody, linked through a disulfide bond to the maytansine derivative DM4) that was used in the treatment of several leukemia cell lines [82]. The activity

with the cell cycle, potentially contributing to effective DAC incorporation [75].

are eligible, bone marrow transplantation is recommended after this therapy.

feature, previous therapeutic history, and drugs applied during previous treatment.

should be studied more intensively in the future [76].

**3.4. Resistance to intensive chemotherapy**

**3.5. Resistance to CD33-targeted therapy**

by ASXL1 gene. [74].

190 Myelodysplastic Syndromes

MDSs represent a very diverse group of hematological malignancies. Treatment of this disease involves several drugs such as LEN, AzaC, and DAC. Unfortunately, the exact mechanism of action is still not fully understood. Therefore, prediction of the response to this treatment is still very complicated. For this reason, a detailed knowledge of the molecular causality of MDS and AML progression will be necessary. Moreover, unclear questions about the molecular mechanisms of these drugs should be answered. Resistance to treatment use for AML and MDS still represents serious problems with causality not fully understood. Research oriented on these topics will bring new knowledge and new predictive molecular markers that will enable the better selection of effective therapy for each patient.

#### **Acknowledgements**

This research was supported by grants from the Slovak APVV grant agency (No. APVV-14-0334) and the VEGA grant agency (Vega 2/0182/13, 2/0028/15, 2/0156/16) and the project: "Diagnostics of socially important disorders in Slovakia, based on modern biotech‐ nologies" ITMS 26240220058, supported by the Research & Developmental Operational Programme funded by the ERDF.

#### **Author details**

Lucia Messingerova1,2, Denisa Imrichova2 , Martina Coculova1 , Marian Zelina2 , Lucia Pavlikova2 , Helena Kavcova2 , Mario Seres2 , Viera Bohacova2 , Boris Lakatos1 , Zdena Sulova2 and Albert Breier1,2\*

\*Address all correspondence to: albert.breier@stuba.sk

1 Institute of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava, Slovakia

2 Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovakia

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### *Edited by Ota Fuchs*

This book is a comprehensive overview of myelodysplastic syndromes (MDS). MDS are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, peripheral cytopenias, frequent karyotypic abnormalities, and risk of transformation to acute myeloid leukemia (AML). Median age of patients with MDS is about 70 years. Various immune abnormalities occur in MDS patients, and the relationship between autoimmune disorders and MDS is described. Accurate prognostication and risk stratification for individual patients with MDS are important for clinical treatment decisions. Patients with MDS are classified into two broad prognostic categories: lower risk and higher risk. The approval of lenalidomide, azacitidine, and decitabine in last 10 years helped to diminish the clinical impact of MDS and delayed its progression to AML.

Myelodysplastic Syndromes

Myelodysplastic Syndromes

*Edited by Ota Fuchs*

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